U.S. patent application number 12/566399 was filed with the patent office on 2010-02-25 for recombinant constructs and transgenic fluorescent ornamental fish therefrom.
Invention is credited to Alan Blake, Richard Crockett, Jeffrey Essner, Perry Hackett, Aidas Nasevicius.
Application Number | 20100050280 12/566399 |
Document ID | / |
Family ID | 39083107 |
Filed Date | 2010-02-25 |
United States Patent
Application |
20100050280 |
Kind Code |
A1 |
Blake; Alan ; et
al. |
February 25, 2010 |
RECOMBINANT CONSTRUCTS AND TRANSGENIC FLUORESCENT ORNAMENTAL FISH
THEREFROM
Abstract
The present invention relates to the method and use of reef
coral fluorescent proteins in making transgenic red, green and
yellow fluorescent zebrafish. Preferably, such fluorescent
zebrafish are fertile and used to establish a population of
transgenic zebrafish and to provide to the ornamental fish industry
for the purpose of marketing. Thus, new varieties of ornamental
fish of different fluorescence colors from a novel source are
developed.
Inventors: |
Blake; Alan; (Austin,
TX) ; Crockett; Richard; (New York, NY) ;
Essner; Jeffrey; (Ames, IA) ; Hackett; Perry;
(Saint Paul, MN) ; Nasevicius; Aidas; (Plant City,
FL) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE., SUITE 2400
AUSTIN
TX
78701
US
|
Family ID: |
39083107 |
Appl. No.: |
12/566399 |
Filed: |
September 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11839364 |
Aug 15, 2007 |
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12566399 |
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60838006 |
Aug 16, 2006 |
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60842721 |
Sep 7, 2006 |
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Current U.S.
Class: |
800/20 ;
800/21 |
Current CPC
Class: |
A01K 2227/40 20130101;
A01K 67/0275 20130101; C07K 14/43595 20130101; C12N 15/8509
20130101; G06Q 99/00 20130101; A01K 2217/052 20130101; A01K 67/027
20130101; A01K 2217/206 20130101; C12N 2830/008 20130101; A01K
2217/05 20130101; A01K 2267/0393 20130101 |
Class at
Publication: |
800/20 ;
800/21 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/00 20060101 C12N015/00 |
Claims
1.-39. (canceled)
40. A method of producing a transgenic fluorescent, ornamental
fish, comprising the steps of: (a) obtaining one or more cloning
vectors, each comprising one or more fluorescent genes under the
control of a promoter, wherein said one or more cloning vectors
does not have a gene encoding antibiotic resistance or a
replication origin; and (b) producing a population of transgenic
fluorescent, ornamental fish wherein one or more fish of said
population comprise one or more fluorescent genes in its genome and
express one or more fluorescent proteins.
41. The method of claim 40, wherein obtaining the cloning vector
comprises preparing a cloning vector and inserting therein one or
more fluorescent genes under the control of a promoter and removing
vector backbone components including gene(s) encoding antibiotic
resistance gene and the replication of origin.
42. The method of claim 40, wherein the transgenic fish is further
defined as a transgenic zebrafish.
43. The method of claim 41, wherein the transgenic zebrafish is
defined as a transgenic Golden zebrafish.
44. The method of claim 40, wherein the transgenic fish has an
integration event at a single locus after selection.
45.-71. (canceled)
72. The method of claim 40, wherein said fish so produced do not
comprise a transgenic antibiotic resistance gene or origin of
replication.
73. A transgenic fluorescent, ornamental fish produced by the
method of claim 40.
74. A transgenic fluorescent, ornamental fish having a genome
comprising one or more fluorescent genes under the control of a
promoter and expressing one or more fluorescent proteins encoded by
said fluorescent genes, and wherein said fish do not comprises a
transgenic antibiotic resistance gene or origin of replication.
75. The transgenic fish of claim 74, wherein the one or more
fluorescent proteins are selected from the group consisting of
ZsGreen1, ZsYellow1, DsRed2, GFP, eGFP, YFP, eYFP, BFP, eBFP, CFP,
eCFP, FP, AmCyan1, DsRed-Express, AsRed2, HcRed1, mPlum, mCherry,
tdTomato, mStrawberry, J-Red, DsRed-monomer, mOrange, mKO,
MCitrine, Venus, Ypet, EYFP, Emerald, CyPet, mCFPm, Cerulean, and
T-Sapphire.
Description
[0001] The present application claims the benefit of previously
filed provisional application Ser. Nos. 60/838,006, filed Aug. 16,
2006, and 60/842,721, filed Sep. 7, 2006, the disclosures of which
are incorporated by reference herein in their entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to transgenic gene constructs with
fish gene promoters and heterologous genes for generation of
transgenic fish, particularly fluorescent transgenic fish.
[0004] 2. Description of Related Art
[0005] Transgenic technology involves the transfer of a foreign
gene into a host organism enabling the host to acquire a new and
inheritable trait. The technique was first developed in mice by
Gordon et al. (1980). They injected foreign DNA into fertilized
eggs and found that some of the mice developed from the injected
eggs retained the foreign DNA. Applying the same technique,
Palmiter et al. (1982) introduced a chimeric gene containing a rat
growth hormone gene under a mouse heavy metal-inducible gene
promoter and generated the first batch of genetically engineered
supermice, which were almost twice as large as non-transgenic
siblings. This work has opened a promising avenue in using the
transgenic approach to provide to animals new and beneficial traits
for livestock husbandry and aquaculture.
[0006] In addition to the stimulation of somatic growth for
increasing the gross production of animal husbandry and
aquaculture, transgenic technology also has many other potential
applications. First, transgenic animals can be used as bioreactors
to produce commercially useful compounds by expression of a useful
foreign gene in milk or in blood. Many pharmaceutically useful
protein factors have been expressed in this way. For example, human
1-antitrypsin, which is commonly used to treat emphysema, has been
expressed at a concentration as high as 35 mg/ml (10% of milk
proteins) in the milk of transgenic sheep (Wright et al., 1991).
Similarly, the transgenic technique can also be used to improve the
nutritional value of milk by selectively increasing the levels of
certain valuable proteins such as caseins and by supplementing
certain new and useful proteins such as lysozyme for antimicrobial
activity (Maga and Murray, 1995). Second, transgenic mice have been
widely used in medical research, particularly in the generation of
transgenic animal models for human disease studies (Lathe and
Mullins, 1993). More recently, it has been proposed to use
transgenic pigs as organ donors for xenotransplantation by
expressing human regulators of complement activation to prevent
hyperacute rejection during organ transplantation (Cozzi and White,
1995). The development of disease resistant animals has also been
tested in transgenic mice (e.g. Chen et al., 1988).
[0007] Fish are also an intensive research subject of transgenic
studies. There are many ways of introducing a foreign gene into
fish, including: microinjection (e.g., Zhu et al., 1985; Du et al.,
1992), electroporation (Powers et al., 1992), sperm-mediated gene
transfer (Khoo et al., 1992; Sin et al., 1993), gene bombardment or
gene gun (Zelenin et al., 1991), liposome-mediated gene transfer
(Szelei et al., 1994), and the direct injection of DNA into muscle
tissue (Xu et al., 1999). The first transgenic fish report was
published by Zhu et al., (1985) using a chimeric gene construct
consisting of a mouse metallothionein gene promoter and a human
growth hormone gene. Most of the early transgenic fish studies have
concentrated on growth hormone gene transfer with an aim of
generating fast growing "superfish". While a majority of early
attempts used heterologous growth hormone genes and promoters and
failed to produce gigantic superfish (e.g. Chourrout et al., 1986;
Penman et al., 1990; Brem et al., 1988; Gross et al., 1992),
enhanced growth of transgenic fish has been demonstrated in several
fish species including Atlantic salmon, several species of Pacific
salmons, and loach (e.g. Du et al., 1992; Delvin et al., 1994,
1995; Tsai et al., 1995).
[0008] The zebrafish, Danio rerio, is a new model organism for
vertebrate developmental biology. As an experimental model, the
zebrafish offers several major advantages such as easy availability
of eggs and embryos, tissue clarity throughout embryogenesis,
external development, short generation time and easy maintenance of
both the adult and the young. Transgenic zebrafish have been used
as an experimental tool in zebrafish developmental biology.
However, for the ornamental fish industry the dark striped
pigmentation of the adult zebrafish does not aid in the efficient
display of the various colors that are currently available in the
market. More recently, Lamason et al. (2005) in their report showed
that the Golden zebrafish carry a recessive mutation in the slc24a5
gene, a putative cation exchanger, and have diminished number, size
and density of melanosomes which are the pigmented organelles of
the melanocytes and hence are lightly pigmented as compared to the
wild type zebrafish. The availability of the Golden zebrafish for
transgenesis with fluorescent proteins would result in better
products for the ornamental fish industry as it would allow for a
better visualization of the various colors.
[0009] Green fluorescent protein (GFP) is a useful tool in the
investigation of various cellular processes. The GFP gene was
isolated from the jelly-fish Aqueous victoria. More recently,
various other new fluorescent protein genes have been isolated from
the Anthozoa class of coral reefs (Matz et al., 1999) called DsRed,
red fluorescent protein gene; ZsGreen, green fluorescent protein
gene and ZsYellow, yellow fluorescent protein gene. The novel
fluorescent proteins encoded by these genes share 26-30% identity
with GFP (Miyawaki, 2002). These are bright fluorescent proteins
and each emits a distinct wavelength. They are physico-chemically
very stable, extremely versatile, emitting strong visible
fluorescence in a variety of cell types and display exceptional
photostability and hence fluoresce over extended periods of time.
Because of their distinct spectra, they can be used in combination.
The crystal structure of the DsRed protein suggests that the
chromofore is located on a central .alpha.-helical segment embedded
within a tightly folded .beta.-barrel and that the DsRed protein
forms tetramers in vivo (Wall et al., 2000).
[0010] Coral reef fluorescent proteins have broad application in
research and development. The red fluorescent protein, DsRed, has
been used as a reporter in the transgenic studies involving various
animal model systems: for example, filamentous fungi (Eckert et
al., 2005, Mikkelsen et al., 2003); ascidian (Zeller et al., 2006);
zebrafish (Zhu et al., 2005, Zhu et al., 2004, Gong et al., 2003,
Finley et al., 2001); xenopus (Werdien et al., 2001); insect (Cho
et al., 2006, Handler et al., 2001, Horn et al., 2002); drosophila
(Barolo et al., 2004); silkworm (Royer et al., 2005); mouse (Schmid
et al., 2006, Vintersten et al., 2004); rat (Sato et al., 2003);
and plants (Wenek et al., 2003). It has also been used a marker in
imaging studies in stem cells (Tolar et al., 2005, Long et al.,
2005) and mouse (Long et al., 2005, Hadjantonakis et al., 2003).
Green fluorescent protein, ZsGreen, has been used as a
transformation marker in insects (Sarkar et al., 2006), knock-in
mouse model for the study of KIT expressing cells (Wouters et al.,
2005) and as reporters for plant transformation (Wenck et al.,
2003). Yellow fluorescent protein, ZsYellow, has been used a
reporter for plant transformation (Wenck et al., 2003) and for
visualizing fungal pathogens (Bourett et al., 2002). All of these
transgenic experiments have aimed at developing newer markers and
reporters for transgenesis; however, progress in the field of
ornamental fish industry has been limited.
SUMMARY OF THE INVENTION
[0011] In certain embodiments, the present invention concerns
making recombinant constructs and transgenic fluorescent fish and
providing such fish to the ornamental fish industry. The term
recombinant construct is used to mean recombinant DNA constructs
having sequences which do not occur in nature or exist in a form
that does not occur in nature or exist in association with other
materials that do not occur in nature. The term transgenic has
historically been used in many contexts with various meanings. In
the embodiments of this invention transgenic is understood to mean
genetic material artificially introduced into the genome of an
organism. An organism incorporating such genetic material, or
progeny to which this genetic material was passed, would be
considered a transgenic organism. Such transgenic organisms may
also, in certain embodiment, be referred to generally as a
genetically modified organism (GMO), which is defined as an
organism whose genetic material has been altered using the genetic
engineering techniques generally known as recombinant DNA
technology. This modified DNA is then transferred into an organism
preferably resulting in the expression of modified or novel traits.
The term "GMO" does not cover organisms whose genetic makeup has
been altered by conventional cross breeding or by "mutagenesis"
breeding, as these methods predate the discovery of the recombinant
DNA techniques. Technically, however, such techniques are by
definition genetic modification. The term fluorescent is used to
mean an entity that absorbs light of one wavelength and emits at a
different wavelength.
[0012] Specific embodiments of the present invention are directed
to methods of making transgenic fluorescent fish having one
sequence from a group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ
ID NO:4 and SEQ ID NO:5, as well as transgenic fish developed by
such methods. Thus, a transgenic zebrafish having integrated into
its germ line cell DNA a transgenic construct comprising one or
more of SEQ ID NO:1 through SEQ ID NO:5 is also included as part of
the invention. Further more, the invention provides transgenic
zebrafish egg and/or sperm cells comprising a sequence according to
SEQ ID NO:1 through SEQ ID NO:5 integrated in its/their genome(s).
In certain aspects of the invention, two or more sequences from a
group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ
ID NO:5 are used in one zebrafish. In a specific aspect, SEQ ID
NO:1 and SEQ ID NO:2 are used in the same fish and SEQ ID NO:3 and
SEQ ID NO:5 are used in the same fish. In preferred embodiments, it
is contemplated that the transgenic fluorescent fish are fertile
transgenic fluorescent fish.
[0013] In another preferred embodiment, the fish for use with the
disclosed constructs and methods is the Golden zebrafish. Zebrafish
skin color is determined by pigment cells in their skin, which
contain pigment granules called melanosomes. The number, size and
density of the melanosomes per pigment cell influence the color of
the fish skin. Golden zebrafish have diminished number, size, and
density of melanosomes and hence have lighter skin when compared to
the wild type zebrafish. Golden zebrafish have a mutation in
slc24a5 gene, rendering the fish skin lighter or less pigmented
(Lamason et al., 2005).
[0014] In another embodiment of the invention, a method for making
transgenic fluorescent fish is provided comprising at least the
following steps: a) preparing a vector which has a transgenic
fluorescence expression cassette comprising one sequence from a
group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ
ID NO:5, or two or more sequences from a group of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 are used in
combination, specifically, SEQ ID NO:1 and SEQ ID NO:2 are used
together and SEQ ID NO:3 and SEQ ID NO:5 are used together; b)
making the transgenic zebrafish using the vectors; and, c)
selecting transgenic zebrafish that fluoresce by monitoring
fluorescence under a light of appropriate wavelength. The
transgenic expression cassette has a set of transcriptional
regulatory motifs, herein referred to as a promoter, which may be
from the host species (herein referred to as a homologous promoter)
or from another species (herein referred to as a heterologous
promoter), heterologous fluorescent gene, and appropriate
RNA-processing and/or translational enhancing motif. The term
promoter as used herein refers to the DNA elements that direct and
regulate transcription. For instance, the zebrafish fast skeletal
muscle myosin light chain promoter and carp .beta.-actin promoter
may be used according to the invention.
[0015] In certain specific embodiments there are provided methods
to use multiple vectors to express at least one fluorescent protein
in order to enhance expression. The preferred mode is to make a
transgenic fish comprising in its genome a first fluorescent
transgene under the control of a ubiquitous fish promoter, and a
second fluorescent transgene under the control of a tissue specific
fish promoter. The ubiquitous fish promoter is selected from the
group consisting of those transcriptional motifs that direct gene
expression in most cells, and more preferably in all cells; they
are also preferably promoters for `housekeeping genes`, such as
tubulin, ribosomal protein, and actin genes. The tissue specific
fish promoter is selected from the group consisting of those
transcriptional motifs that are active in specific cells of
differentiated tissues such as muscle, brain, liver, blood and
eyes. In a preferred embodiment, the tissue specific fish promoter
is muscle specific. As used herein, a promoter drives expression
"specifically" in a tissue if the level of expression is at least
5-fold, preferably at least 10-fold higher, more preferably at
least 50-fold higher in that tissue than in any other tissue.
[0016] More than one construct can be injected into the fish
embryos simultaneously. For example, in the present invention, both
Red zebrafish 1 and Green zebrafish 1 incorporate more than one
transgenic expression cassettes, with one being a ubiquitous
promoter, and the other being a strong muscle promoter. In
particular, Red zebrafish 1 incorporates the cassettes represented
by FIG. 1 and FIG. 4, and Green zebrafish 1 incorporates the
cassettes represented by FIG. 2 and FIG. 5. While the present
invention incorporates only the transgenic insert cassettes shown
in the Figures, it is understood that multiple transgenic insert
cassettes of any type can be simultaneously injected into a fish
embryo from any species.
[0017] The steps involved in making the transgenic fish further
involve isolation and separation of the transgenic expression
cassette from the vector backbone to remove any gene encoding
antibiotic resistance (e.g., ampicillin or kanamycin) and origin of
replication. In a preferred mode, a suitable promoter would be
expected to drive stable and consistent expression throughout the
life of the fish. To achieve such stable expression, it is
necessary to choose a promoter that is known to drive stable and
consistent expression throughout the life of the fish. For example,
a promoter that drives expression only during the six months of the
life of the fish would not be suitable for use. Examples of
suitable promoters may be selected from the group consisting of
those for housekeeping genes, such as tubulin, ribosomal protein,
and actin gene promoters.
[0018] It is also preferred to use regulatory elements, for
example, RNA processing and translational enhancing elements in the
transgenic insert cassette to produce a transgenic fluorescent,
ornamental fish. The RNA processing signals, preferably, are one or
more polyadenylation signals and/or one or more introns. Since
introns are sequences between exons, the presence of an intron
automatically indicates the presence of two exons. Accordingly, two
introns indicate the presence of three exons, and so on. The carp
beta-actin intron used in SEQ ID 2 and SEQ ID 5 is an example of
such an intron, and the untranslated carp beta-actin exon used in
SEQ ID 2 and SEQ ID 5 is an example of such an exon. Exons and
introns other than carp beta-actin can be used as well. The
translational enhancing elements, preferably, are 5' untranslated
leader sequences of 40-200 nucleotides, and more preferably
untranslated leader sequences of 40-70 nucleotides. It is known
that the presence of introns in primary transcripts can increase
expression, possibly by causing the transcript to enter the
processing and transport system for mRNA. It is also preferred that
the intron be homologous to the host species, and more preferably
homologous to the expression sequences used (that is, that the
intron be from the same gene that some or all of the expression
sequences are from).
[0019] The disclosed transgene constructs preferably include other
sequences which improve expression from, or stability of, the
construct. For example, including a polyadenylation signal on the
constructs encoding a protein ensures that transcripts from the
transgene will be processed and transported as mRNA. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that efficient
polyadenylation signals, such as those derived from viruses, be
used in the transgenic constructs, and more preferred to use at
least two polyadenylation signals, which more preferably are two
copies of SV40 polyadenylation sequence.
[0020] It is also a subject of this invention to disclose
expression of the fluorescent protein gene only in chromatophores.
There are several types of chromatophores found in animals:
melanophores (black), xanthophores (yellow), erythrophores (red),
cyanophores (blue), leucophores (white) and iridophores
(reflective). Different species of fish contain all types of
chromatophores, usually a subset of them in different combinations.
Zebrafish contain melanophores, xantophores and iridophores. These
different cell types express specific genes, characteristic only
for them or specific for a subset of chromatophores. In a preferred
embodiment, promoters of these specific genes fused to fluorescent
protein open reading frames (ORFs) can be used to visualize
specific chromatophores. The specific genes can be roughly divided
into two major groups: regulatory proteins and biosynthesis
enzymes, involved in specific pigment synthesis. Expression of
regulatory proteins usually is at lower level than that of
biosynthesis enzymes therefore use of promoters of biosynthesis
enzymes are most preferred.
[0021] The heterologous fluorescent gene may be, for example, a
gene encoding DsRed2, ZsGreen1 and ZsYellow1. The heterologous
fluorescent gene may also be any variation or mutation of these
genes, encoding fluorescent proteins including green fluorescent
protein (GFP), enhanced green fluorescent protein (eGFP), yellow
fluorescent protein (YFP), enhanced yellow fluorescent protein
(eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent
protein (eBFP), cyan fluorescent protein (CFP) and enhanced cyan
fluorescent protein (eCFP) or any of the proteins listed in Table
4, or any variation or mutation thereof, or any other fluorescence
proteins. The steps involved in making the transgenic fish also
involve introduction of the transgenic expression cassette into the
zebrafish embryos or zebrafish embryonic stem cells. Such embryos
and cells are allowed to grow and mature into adult fish and then
they are screened for the presence of the transgenic expression
cassette using the various molecular biology methods described in
the detailed description section and/or by functional biochemical
assays such as assaying for the activity of the introduced
fluorescent gene by exposing the said fish to light of appropriate
wavelength and/or by visibly inspecting the fish and observing the
expression. Transgenic fluorescent fish are further bred to insure
transmission of the transgenic expression cassette via the germ
cells of a fish as further described in this application.
[0022] The present invention also provides a method to obtain a
progenitor of a new line of fluorescent transgenic fish, and a
population therefrom, which exhibit strong visible fluorescence.
Strong visible fluorescence means that a person with 20/20 vision
(i.e., average vision) will be able to distinguish between the
fluorescent fish in question and a non-fluorescent fish of the same
species at a distance of at least 5 feet in a lighted office, with
a preferred distance of at least 10 feet in a lighted office, and a
more preferred distance of at least 15 feet in a lighted office,
and an even more preferred distance of at least 20 feet in a
lighted office, with the illumination level defined in Table 5. One
can observe all transgenic fluorescent fish from a particular
population that exhibit strong visible fluorescence under the
various lighting conditions and select the fish that exhibits the
highest level of visible fluorescence of the fluorescent protein.
Selected fish with strong visible fluorescence are monitored and
their progeny selected continuously to ensure stability of
expression and maintenance of strong visible fluorescence. Thus a
new line of fish that exhibit strong visible fluorescence is
created for further breeding.
[0023] Transgenic fish made by the present disclosure will emit
red, yellow-green and yellow-orange fluorescence under light of
distinct wavelengths and hence will be unique and attractive to the
ornamental fish industry. In yet another embodiment of the
invention, a method of making the transgenic fish available to the
consumer by a grower or a commercial distributor through a retailer
for sale to the public. In such embodiment, the fish may also be
sold by the grower or commercial distributor to a regional
wholesale distributor, who will then sell to a retailer for sale to
the public. The fluorescent transgenic fish are also useful for the
development of a biosensor system and as research models for
embryonic studies such as cell lineage, cell migration, cell and
nuclear transplantation, cell-cell interaction in vivo, etc.
[0024] Transgenic zebrafish comprising an expression cassette
according to the invention may be homozygous or heterozygous with
respect to the expression cassette. In some preferred aspects, fish
for use in breeding of transgenic zebrafish of the invention will
be homozygous for an expression cassette. Homozygous fish bred with
fish lacking an expression cassette (e.g., Golden zebrafish) will
in nearly all cases produce 100% heterozygous offspring. Likewise,
transgenic fish for commercial retail will preferably be
heterozygous for an expression cassette. Furthermore in some very
specific aspects a transgenic fish of the invention comprises the
specific integration event of the Red fluorescent expression
cassette described in Example 3.
[0025] In certain specific embodiments there are provided
transgenic fluorescent zebrafish comprising specific transgenic
integration events. These fish are of particular interest since,
for example, they embody an esthetically pleasing level of protein
fluorescence. Thus, in some embodiments there is provided a
transgenic zebrafish comprising a chromosomally integrated
expression cassette encoding a DsRed2 gene wherein the zebrafish
comprises the Red zebrafish 1 transformation event, sperm
comprising said Red zebrafish 1 transformation event having been
deposited as ECACC accession no. 06090403. In some other aspects,
there is provided a transgenic zebrafish comprising a chromosomally
integrated expression cassette encoding a ZsGreen1 gene wherein the
zebrafish comprises the Green zebrafish 1 transformation event,
sperm comprising said Green zebrafish 1 transformation event having
been deposited as ECACC accession no. 06090401. In still other
aspects, there is provided a transgenic zebrafish comprising a
chromosomally integrated expression cassette encoding a ZsYellow1
gene wherein the zebrafish comprises the Yellow zebrafish 1
transformation event, sperm comprising said Yellow zebrafish 1
transformation event having been deposited as ECACC accession no.
06090402. As described above, transgenic fish comprising these
specific transgenic events may be homozygous or heterozygous for
transgene, and in some cases may comprise more than one of the
transgenic events, although it is preferred to have only one
integration location for any given transgenic modification. Eggs,
sperm and embryos comprising these specific transgenic events are
also included as part of the instant invention.
[0026] Any of the fluorescence genes noted in this application may
be used in similar embodiments of this invention. Embodiments
discussed in the context of a method and/or composition of the
invention may be employed with respect to any other method or
composition described herein. Thus, an embodiment pertaining to one
method or composition may be applied to other methods and
compositions of the invention as well.
[0027] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0028] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0029] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0030] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The following drawings are part of the present specification
and are included to further demonstrate certain aspects of the
present invention. The invention may be better understood by
reference to the drawing in combination with the detailed
description of specific embodiments presented herein.
[0032] FIG. 1: The figure shows a schematic map of the transgenic
construct, pZMLC-DsRed2-SV40x2. The 2.1-kb eukaryotic promoter
sequence zMLC-1934 promoter was amplified by PCR from pMLC vector
and cloned into XhoI and EcoRI restriction sites. The 684 bp DsRed2
fluorescent protein CDS was amplified by PCR from pDsRed2-N1
(Clontech) and inserted into EcoRI and SpeI sites. The 440-bp
3'UTR/poly(A) sequence encoding tandem SV40 polyadenylation signals
was PCR amplified from pK-SV40(A)x2 and cloned into SpeI and NotI
sites. XhoI, XmnI and NotI restriction sites were used to isolate
the expression construct from the vector backbone. Also shown is
the ampicillin (Amp, formally known as beta-lactamase (bla))
resistance gene in the backbone of the pBluescript plasmid. The
total length of the recombinant plasmid pzMLC-DsRed2-SV40x2 is 6009
bp.
[0033] FIG. 2: The figure shows a schematic map of the transgenic
construct, pZMLC-ZsGreen1-SV40x2. The 1.9-kb eukaryotic promoter
sequence zMLC-1934 promoter was amplified by PCR from pMLC vector
and cloned into XhoI and EcoRI restriction sites. The 716 bp
ZsGreen1 fluorescent protein CDS was amplified by PCR from
pZsGreen1-N1 (Clontech) and inserted into EcoRI and SpeI sites. The
440-bp 3'UTR/poly(A) sequence encoding tandem SV40 polyadenylation
signals was PCR amplified from pK-SV40(A)x2 and clone into SpeI and
NotI sites. XhoI, XmnI and NotI restriction sites were used to
isolate the expression construct from the vector backbone. Also
shown is the ampicillin (Amp) resistance gene in the backbone of
the pBluescript plasmid. The total length of the recombinant
plasmid pzMLC-ZsGreen1-SV40x2 is 6041 bp.
[0034] FIG. 3: The figure shows is a schematic map of the
transgenic construct, pZMLC-ZsYellow1-SV40x2. The 1.9-kb eukaryotic
promoter sequence zMLC-1934 promoter was amplified by PCR from pMLC
vector and cloned into XhoI and EcoRI restriction sites. The 718 bp
ZsYellow1 fluorescent protein CDS was amplified by PCR from
pZsYellow1-N1 (Clontech) and inserted into EcoRI and SpeI sites.
The 440-bp 3'UTR/poly(A) sequence encoding tandem SV40
polyadenylation signals was PCR amplified from pK-SV40(A)x2 and
clone into SpeI and NotI sites. XhoI, XmnI and NotI restriction
sites were used to isolate the expression construct from the vector
backbone. Also shown is the ampicillin (Amp) resistance gene in the
backbone of the pBluescript plasmid. The total length of the
recombinant plasmid pzMLC-ZsYellow1-SV40x2 is 6043 bp.
[0035] FIG. 4: The figure shows a schematic map of the transgenic
construct, pCBAC-DsRed2-SV40x2. The 2.5-kb common carp beta-actin
enhancer/promoter sequence, beta-actin exon-1 and beta-actin
intron-1 was amplified by PCR from pFV7b vector and cloned into
XbaI and KpnI restriction sites. The 684 bp DsRed2 fluorescent
protein CDS was amplified by PCR from pDsRed2-N1 (Clontech) and
inserted into EcoRI and SpeI sites. The 443-bp 3'UTR/poly(A)
sequence encoding tandem SV40 polyadenylation signals sequence
encoding tandem SV40 signal was PCR amplified from pK-SV40(A)x2 and
cloned into SpeI and AatII sites. XbaI and AatII restriction sites
were used to isolate the expression construct from the vector
backbone. Also shown is the ampicillin (Amp) resistance gene in the
backbone of the pBluescript plasmid. The total length of the
recombinant plasmid pCBAC-DsRed2-SV40x2 is 5801 bp.
[0036] FIG. 5: The figure shows a schematic map of the transgenic
construct, pCBAC-ZsGreen1-SV40x2. The 2.5-kb carp beta-actin
enhancer/promoter sequence, beta-actin exon 1 and beta-actin intron
1 was amplified by PCR from pFV7b vector and cloned into XbaI and
KpnI restriction sites. The 716 bp ZsGreen1 fluorescent protein CDS
was amplified by PCR from pZsGreen1-N1 (Clontech) and inserted into
EcoRI and SpeI sites. The 443 bp 3'UTR/poly(A) sequence encoding
tandem SV40 polyadenylation signals sequence encoding tandem SV40
signal was PCR amplified from pK-SV40(A)x2 and cloned into SpeI and
AatII sites. XbaI and AatII restriction sites were used to isolate
the expression construct from the vector backbone. Also shown is
the ampicillin (Amp) resistance gene in the backbone of the
pBluescript plasmid. The total length of the recombinant plasmid
pCBAC-ZsGreen1-SV40x2 is 5833 bp.
[0037] FIG. 6: Transgenic Construct purification and injection
process. The Figure depicts step by step the process of transgenic
construct purification and injection. Step 1 illustrates separation
of the plasmid backbone sequence with the antibiotic resistance
gene and origins of replication (pUC ori and f1(-) ori) (on left)
and the expression construct (on right). Step 2 and 3 show the
method of purification of the expression construct by loading and
electrophoretic separation of the DNA fragments on an agarose gel.
The antibiotic resistance gene and origins of replication (pUC ori
and f1(-) ori) are below the expression construct on the gel. Step
4 exemplifies the process of microinjection of the gel-purified
expression construct in to the fertilized zebrafish embryos.
DETAILED DESCRIPTION OF THE INVENTION
Transgenic Constructs
[0038] The present invention encompasses transgenic constructs
which are genetic material artificially introduced into fish to
produce a transgenic fish. The manner of introduction, and, often,
the structure of a transgenic construct, render such a transgenic
construct an exogenous construct. Although a transgenic construct
can be made up of any assembly of nucleic acid sequences, for use
in the disclosed transgenic fish it is preferred that the
transgenic constructs combine regulatory elements operably linked
to a sequence encoding one or more proteins. The methods and
protocols for designing and making transgenic constructs are well
known to those skilled in the art and can be found, for example, in
Sambrook et al., 2001; Sambrook et al., 1989 and U.S. Pub No.
2004/0143864 A1, all of which are hereby incorporated by reference
in their entireties.
[0039] To develop successful transgenic fish with a predictable
pattern of transgenic expression, the first step is to make the
appropriate genetic construct. The genetic construct generally
comprises three portions: transcriptional regulators comprising a
promoter, a gene and appropriate RNA-processing and/or
translational enhancing motif. The gene promoter would determine
where, when and under what conditions the gene is expressed. The
gene contains protein coding portions that determine the protein to
be synthesized and thus the biological function. The gene might
also contain intron sequences which can affect mRNA processing or
which might contain transcription regulatory elements. The RNA
processing signals may include: one or more polyadenylation signals
and one or more introns. Among the three portions, it is preferable
to use a promoter that drives strong expression. The promoter may
be a homologous promoter or it may be a heterologous promoter.
[0040] A promoter drives expression "predominantly" in a tissue if
expression is at least 2-fold, preferably at least 5-fold higher in
that tissue compared to a reference tissue. A promoter drives
expression "specifically" in a tissue if the level of expression is
at least 5-fold, preferably at least 10-fold higher, more
preferably at least 50-fold higher in that tissue than in any other
tissue. A ubiquitous promoter drives expression in most tissues,
and preferably in all tissues.
Recombinant DNA Constructs
[0041] Recombinant DNA constructs comprising one or more of the DNA
sequences described herein and an additional DNA sequence are also
included within the scope of this invention. These recombinant DNA
constructs usually have sequences which do not occur in nature or
exist in a form that does not occur in nature or exist in
association with other materials that do not occur in nature. The
DNA sequences described as constructs or in vectors above are
"operably linked" with other DNA sequences. DNA regions are
operably linked when they are functionally related to each other.
Generally, operably linked means contiguous (or in close proximity
to).
[0042] The disclosed transgenic constructs preferably include other
sequences that improve expression from, or stability of, the
construct. For example, including a polyadenylation signal on the
constructs encoding a protein ensures that mRNA transcripts from
the transgene will be efficiently translated as protein. The
identification and use of polyadenylation signals in expression
constructs is well established. It is preferred that defined and
efficient polyadenylation signals, such as those derived from
viruses, be used in the transgenic constructs, and more preferred
to use at least two polyadenylation signals, which more preferably
are two copies of SV40 polyadenylation sequence.
[0043] In certain specific embodiments there are provided methods
to use multiple vectors to express at least one fluorescent protein
in order to enhance expression. The preferred mode is to make a
transgenic fish comprising in its genome a first fluorescent
transgene under the control of a ubiquitous fish promoter, and a
second fluorescent transgene under the control of a tissue specific
fish promoter. In a preferred embodiment, the tissue specific fish
promoter is muscle specific. The ubiquitous fish promoter and the
muscle specific promoter are, for example, selected from Table 1
below. In the Table 1, any promoter marked with an "X" is confirmed
available at this time, with any unmarked promoter, or any other
promoter of interest, available preferably through the following
steps: following the database searching instructions provided in
detail below, conducting a literature search, and sequencing the
gene and promoter of interest through methods that are well know by
artisans in the field.
[0044] The provided Table 1 of muscle-specific and ubiquitous
promoters constitutes only a small portion of publicly available
promoters. An extensive list of genes with expression of interest
(e.g., muscle-specific expression) can be found using NCBI protein
database server (www at ncbi.nlm.nih.gov/sites/entrez?db=Protein).
For example, in order to find mouse genes expressed in skeletal
muscles a search string "mouse skeletal muscle" can be used. The
search results in a list of proteins including their accession
number (e.g., CAA47621) and their name (e.g., mouse fast skeletal
muscle SR calcium ATPase). In order to find genome information
(e.g., sequence) of the found proteins, Ensembl Genome Browser (www
at ensembl.org/index.html) can be employed, using the accession
number ((e.g., CAA47621) as a search string. The search output will
yield Ensembl gene ID (e.g., ENSMUSG00000030730), gene homologues
in other organisms (e.g., zebrafish--Danio rerio), as well as
genomic information of the gene of interest, including genomic
sequence of the coding region (introns and exons), as well as
genomic DNA sequence surrounding the coding sequence (e.g., "[Exon
info]" link directs a user to the sequence information). Gene
promoters are located upstream (5' flanking sequence) from the
coding sequence, often within several (e.g., five) kilobases. In
addition, some regulatory sequences can be found in introns of the
gene of interest--these regulatory sequences are usually omitted
from constructing tissue-specific gene expression "drivers" due to
complexity of locating them. It is to be understood that the same
approach can be used starting with "zebrafish skeletal muscle" or
"medaka skeletal muscle" or any other species. The searcher may
then continue the search as suggested above to find the genome and
promoter information of interest. It is also to be understood that
methods similar to the one described for searching the database
referenced above can be used to search other existing sequence
databases, as well as databases that are likely to be compiled in
the future.
TABLE-US-00001 TABLE 1 Muscle specific and ubiquitous promoters for
fish expression Gene promoter Zebrafish Fugu Tetraodon Medaka
Xenopus l Rat Mouse Dog Bovine Muscle specific Muscle creatine
kinase X X X X X X X X X MyoD X X X X X X X X X Myogenin X X X X X
X X X X Desmin X X X X X X Muscle enolase-3 X X X X X
beta-sarkoglycan X X X X X X X X X Dystrophin X X X X X X X X Serum
response factor X X X X X X X X X a-tropomyosin X X X X X X X X X
Myosin heavy chain X X X X X X X X Mitochondrial creatine kinase 2
X X X X X X X Myosin light chain X X X X X X X X X Ca2+
transporting ATPase(fast twitch 1) X X skeletal Troponin T1(slow) X
X Tropomodulin 4 X X Four and a half LIM domains 1 X X Fast-type
myosin binding protein C X X Calsequestrin 1 X X Fast muscle
troponin C2 X X Phosphorylase kinase alpha 1 X X Skeletal troponin
I(fast 2) X X Ubiquitous EF-1 alpha X X X X X X X X X Histone 2A ZA
X X X X X X X Acidic ribosomal phosphoprotein PO (ARP) X X X X X X
alpha-catenin X X X X X X X X X beta-catenin X X X X X X X X
gamma-catenin X X X X X X X X X Srb7 X X X X X X X Creatine
kinase(mitochondrial 1) X X Ubiquitous Ca2+ transporting ATPase X X
Ancient ubiquitous protein X X Ubiquitin specific peptidase 4 X X
Acetyl-Coenzyme A acryltransferase 2 X X Monoglyceride lipase X X
Splicing factor 3b subunit 1 X X Tubulin .beta.5 X X Beta-Actin
[0045] Table 2, below, is a partial list of Ensembl gene ID numbers
of mouse and zebrafish skeletal muscle specific and ubiquitous
genes found using this approach.
TABLE-US-00002 TABLE 2 Ensembl IDs of Muscle Specific Promoters
MUSCLE CREATINE KINASE PROMOTERS Zebrafish: (ENSDARG00000035327)
Fugu: (SINFRUG00000143294) Tetraodon: (GSTENG00012956001) Medaka:
(ENSORLG00000000449) Xenopus tropicalis: (ENSXETG00000019108) Rat:
(ENSRNOG00000016837) Mouse: (ENSMUSG00000030399) Dog:
(ENSCAFG00000004507) Bovine: (ENSBTAG00000013921) MYOD PROMOTERS
Zebrafish: (ENSDARG00000030110) Fugu: (SINFRUG00000154785)
Tetraodon: (GSTENG00003954001) Medaka: (ENSORLG00000000694) Xenopus
tropicalis: (ENSXETG00000001320) Rat: (ENSRNOG00000011306) Mouse:
(ENSMUSG00000009471) Dog: (ENSCAFG00000009066) Bovine:
(ENSBTAG00000002216) MYOGENIN PROMOTERS Zebrafish:
(ENSDARG00000009438) Fugu: (SINFRUG00000121801) Tetraodon:
(GSTENG00013986001) Medaka: (ENSORLG00000015906) Xenopus
tropicalis: (ENSXETG00000001704) Rat: (ENSRNOG00000030743) Mouse:
(ENSMUSG00000026459) Dog: (ENSCAFG00000010309) Bovine:
(ENSBTAG00000006030) DESMIN PROMOTERS Zebrafish:
(ENSDARG00000058656) Fugu: (SINFRUG00000121939) Xenopus tropicalis:
(ENSXETG00000019275) Rat: (ENSRNOG00000019810) Mouse:
(ENSMUSG00000026208) Dog: (ENSCAFG00000015475) Bovine:
(ENSBTAG00000005353) MUSCLE ENOLASE 3 BETA PROMOTERS Zebrafish:
(ENSDARG00000039007) Tetraodon: (GSTENG00003809001) Rat:
(ENSRNOG00000004078) Mouse: (ENSMUSG00000060600) Bovine:
(ENSBTAG00000005534) BETA-SARCOGLYCAN PROMOTERS Zebrafish:
(ENSDARG00000052341) Fugu: (SINFRUG00000123612) Tetraodon:
(GSTENG00032779001) Medaka: (ENSORLG00000000171) Xenopus
tropicalis: (ENSXETG00000011676) Rat: (ENSRNOG00000002135) Mouse:
(ENSMUSG00000029156) Dog: (ENSCAFG00000002001) Bovine:
(ENSBTAG00000014601) DYSTROPHIN PROMOTERS Zebrafish:
(ENSDARG00000008487) Fugu: (SINFRUG00000144815) Tetraodon:
(GSTENG00024870001) Medaka: (ENSORLG00000020638) Xenopus
tropicalis: (ENSXETG00000012391) Rat: (ENSRNOG00000003667) Mouse:
(ENSMUSG00000045103) Bovine: (ENSBTAG00000008254) SERUM RESPONSE
FACTOR PROMOTERS Zebrafish: (ENSDARG00000053918) Fugu:
(SINFRUG00000162928) Tetraodon: (GSTENG00025109001) Medaka:
(ENSORLG00000013036) Xenopus tropicalis: (ENSXETG00000018511) Rat:
(ENSRNOG00000018232) Mouse: (ENSMUSG00000015605) Dog:
(ENSCAFG00000001829) Bovine: (ENSBTAG00000012777) ALPHA-TROPOMYOSIN
PROMOTERS Zebrafish: (ENSDARG00000033683) Fugu:
(SINFRUG00000130484) Tetraodon: (GSTENG00015950001) Medaka:
(ENSORLG00000012326) Rat: (ENSRNOG00000018184) Mouse:
(ENSMUSG00000032366) Dog: (ENSCAFG00000016966) Bovine:
(ENSBTAG00000005373) MYOSIN HEAVY CHAIN PROMOTERS Zebrafish:
(ENSDARG00000035437) Fugu: (SINFRUG00000135173) Medaka:
(ENSORLG00000001985) Xenopus tropicalis: (ENSXETG00000023939) Rat:
(ENSRNOG00000031400) Mouse: (ENSMUSG00000033196) Dog:
(ENSCAFG00000023926) Bovine: (ENSBTAG00000007090) MITOCHONDRIAL
CREATINE KINASE (SARCOMERIC, CKMT2) PROMOTERS Zebrafish:
(ENSDARG00000035079) Fugu: (SINFRUG000000160265) Tetraodon:
(GSTENG00028607001) Medaka: (ENSORLG00000000769) Mouse:
(ENSMUSG00000021622) Dog: (ENSCAFG00000008707) Bovine:
(ENSBTAG00000001003) MYOSIN LIGHT CHAIN PROMOTERS Zebrafish:
(ENSDARG00000017441) Fugu: (SINFRUG00000125026) Tetraodon:
(GSTENG00015855001) Medaka: (ENSORLG00000015981) Xenopus
tropicalis: (ENSXETG00000006917) Rat: (ENSRNOG00000013262) Mouse:
(ENSMUSG00000061816) Dog: (ENSCAFG00000013875) Bovine:
(ENSBTAG00000009707)
[0046] While this approach will result in a great number of
sequences, additional points should be considered to generate a
list of strong promoters. For example, abundant structural genes
(e.g., myosin) or abundant enzymes (e.g., SR calcium ATPase) are
likely to yield strong promoters. This screening can easily be
performed by an artisan in the field.
[0047] Preferably more than one construct with different promoters
can be injected into the fish embryos simultaneously. For example,
in the present invention, both Red zebrafish 1 and Green zebrafish
1 incorporate more than one transgenic expression cassette, with
one being a ubiquitous promoter, and the other being a strong
muscle promoter. In particular, Red zebrafish 1 incorporates the
cassettes represented by FIG. 1 and FIG. 4, and Green zebrafish 1
incorporates the cassettes represented by FIG. 2 and FIG. 5. While
the present invention incorporates only the transgenic insert
cassettes shown in the Figures, it is understood that multiple
transgenic insert cassettes of any type can be simultaneously
injected into a fish embryo from any species.
[0048] It is also a subject of this invention to disclose
expression of the fluorescent protein gene specifically in
chromatophores. Chromatophores are pigment-containing and
light-reflecting cells found in animals. There are several types of
chromatophores: melanophores (black), xanthophores (yellow),
erythrophores (red), cyanophores (blue), leucophores (white) and
iridophores (reflective). Of those, only melanophores, called
melanocytes, are found in higher vertebrates, such as mammals.
Different species of fish contain all types of chromatophores,
usually a subset of them in different combinations. Zebrafish
contain melanophores, xantophores and iridophores. These different
cell types express specific genes, characteristic only for them or
specific for a subset of chromatophores. For example,
tyrosinase-related protein 1 (tyrp1) is found only in melanophores;
ednrb1 is found in malenocytes and iridophores. Promoters of these
specific genes fused to fluorescent protein open reading frames
(ORFs) can be used to visualize specific chromatophores. For
example, fugu tyrp1 promoter can be used to drive fluorescent
protein expression in melanophores in zebrafish. The specific genes
can be roughly divided into two major groups: regulatory proteins
(for example, kit--a receptor tyrosine kinase, specific to
melanophores) and biosynthesis enzymes, involved in specific
pigment synthesis (for example, sepiapterin reductase, involved in
yellow pigment synthesis in xanthophores). Expression of regulatory
proteins usually is at lower level than that of biosynthesis
enzymes therefore use of promoters of biosynthesis enzymes are most
preferred. A chromatophore-specific gene expression is outlined in
Table 3 below.
[0049] Of all chromatophores, melanophores have been studied most
extensively (due to their relevance to human biology). Therefore, a
lot is known about transcription factors specific to melanophores,
as well as biosynthesis enzymes involved in melanin synthesis in
different classes of organisms, ranging from lower vertebrates to
humans. The next best characterized chromatophores are the
Xanthophores, for which a number of genes have been isolated,
yielding, a number of known promoters to choose from. With respect
to iridophores, a few specific genes have been isolated (for
example, endothelin receptor b1 Ednrb1). The least known
chromatophores are the cyanophores--neither the nature of their
pigment, nor specification pathway of the cells per se is
known.
TABLE-US-00003 TABLE 3 Chromatophore-specific expressed genes in
fishes Chromatophore Protein Synth/Reg Organism Reference
Iridophore ednrb1 Reg Zebrafish Parichy et al, Developmental
(endothelin Biology 227, 294-306 (2000) receptor b1) Xanthophore
xanthine Synth Guppy Ben et al, Mar Biotechnol dehydrogenase
(Poecilia (NY). 2003 Nov-Dec; 5(6): reticulata); 568-78. Epub 2003
Aug 21; Zebrafish Parichy et al, Developmental Biology 227, 294-306
(2000) sepiapterin Synth medaka Negishi et al, Pigment Cell
reductase (Oryzias Res. 2003 Oct; 16(5): 501-3 latipes) Xanthine
Synth oxidoreductase Fms/Csfl Reg zebrafish Ziegler, Pigment Cell
Res. 2003 Jun; 16(3): 172-82; Ziegler et al, J Biol Chem. 2000 Jun
23; 275(25): 18926-32; Parichy et al, Development 127, 3031-3044
(2000) Melanophores Mitf Reg Zebrafish kit Reg Zebrafish tyrp1
Synth zebrafish, Zou et al, Pigment Cell Res. fugu 2006 Dec; 19(6):
615-27 tyrosinase Synth rana Miura et al, Jpn J Genet.
nigromaculata 1995 Feb; 70(1): 79-92 tyrosinase Synth medaka
Inagaki et al, Pigment Cell Res. 1998 Oct; 11(5): 283-90 tyrosinase
Synth Mouse in Matsumoto et al, Pigment medaka Cell Res. 1992 Nov;
5(5 Pt 2): 322-7 trp2 (tyrosinase- Synth mouse Zhao & Over
beek, Dev Biol. related protein 2) 1999 Dec 1; 216(1): 154-63
dopachrome Synth tautomerase
[0050] It is also known that the presence of introns in primary
transcripts can increase expression, possibly by causing the
transcript to enter the processing and transport system for mRNA.
It is preferred that the intron be homologous to the host species,
and more preferably homologous to the expression sequences used
(that is, that the intron be from the same gene that some or all of
the expression sequences are from). The use and importance of these
and other components useful for transgenic constructs are discussed
in Palmiter et al. (1991); Sippel et al. (1992); Kollias and
Grosveld (1992); and Clark et al. (1993).
[0051] The steps involved in making the transgenic fish further
involve isolation and separation of the transgenic expression
cassette from the vector backbone to remove the gene encoding
antibiotic (e.g., ampicillin or kanamycin) resistance and origin of
replication. In a preferred mode, a suitable promoter is chosen
which is expected to drive stable expression throughout the life of
the fish. To achieve such stable expression, it is necessary to
choose a promoter that is known to drive stable and consistent
expression throughout the life of the fish. For example, a promoter
that drives expression only during the six months of the life of
the fish would not be suitable to achieve stable expression
throughout the life of the fish.
[0052] The heterologous fluorescent gene may be, for example, a
gene encoding DsRed2, ZsGreen1 and ZsYellow1. The heterologous
fluorescent gene may also be any variation or mutation of these
genes, encoding fluorescent proteins including green fluorescent
protein (GFP), enhanced green fluorescent protein (eGFP), yellow
fluorescent protein (YFP), enhanced yellow fluorescent protein
(eYFP), blue fluorescent protein (BFP), enhanced blue fluorescent
protein (eBFP), cyan fluorescent protein (CFP) and enhanced cyan
fluorescent protein (eCFP) or any of the proteins listed in Table
4, below, or any variation or mutation thereof, or any other
fluorescence proteins. The steps involved in making the transgenic
fish also involve introduction of the transgenic expression
cassette into the zebrafish embryos or zebrafish embryonic stem
cells. Such embryos and cells are allowed to grow and mature into
adult fish and then they are screened for the presence of the
transgenic expression cassette using the various molecular biology
methods described in the detailed description section and/or by
functional biochemical assays such as assaying for the activity of
the introduced fluorescent gene by exposing the said fish to light
of appropriate wavelength and/or by visibly inspecting the fish and
observing the expression. Transgenic fluorescent fish are further
bred to insure transmission of the transgenic expression cassette
to the germ cells of a fish as further described in this
application.
TABLE-US-00004 TABLE 4 Fluorescent Proteins ("FP") with their
Maximum Excitation and Emission Wavelengths FP Excitation max (nm)
Emission max (nm) AmCyan1 458 489 ZsGreen1 493 505 ZsYellow1 529
539 DsRed2 563 582 DsRed-Express 557 579 AsRed2 576 592 HcRed1 588
618 mPlum 590 649 mCherry 587 610 tdTomato 554 581 mStrawberry 574
596 J-Red 584 610 DsRed-monomer 556 586 mOrange 548 562 mKO 548 559
MCitrine 516 529 Venus 515 528 Ypet 517 530 EYFP 514 527 Emerald
487 509 EGFP 488 507 CyPet 435 477 mCFPm 433 475 Cerulean 433 475
T-Sapphire 399 511
[0053] The sequences of the DNAs which are useful in the invention
are set forth in the attached Sequence Listing.
[0054] The sequence listed herein as SEQ ID NO:1 is the transgenic
fluorescence expression cassette having zebrafish fast skeletal
muscle specific myosin light chain (zMLC) promoter, DsRed2 (a red
fluorescent protein gene from Anthozoa, a reef coral), and two
copies of the SV40 polyadenylation sequence. The sequence listed in
SEQ ID NO:1 is the complementary sequence to the coding DNA
strand.
[0055] The sequence listed herein as SEQ ID NO:2 is the transgenic
fluorescence expression cassette having carp ubiquitous
.beta.-actin enhancer/promoter, DsRed2 (a red fluorescent protein
gene from Anthozoa, a reef coral), and two copies of the SV40
polyadenylation sequence. The first exon and intron of .beta.-actin
has been incorporated in the SEQ ID NO:2 to allow for increased
expression of the fluorescence protein gene.
[0056] The sequence listed herein as SEQ ID NO:3 is the transgenic
fluorescence expression cassette having zebrafish fast skeletal
muscle specific myosin light chain (zMLC) promoter, ZsGreen1 (a
green fluorescent protein gene from Anthozoa, a reef coral), and
two copies of the SV40 polyadenylation sequence. The sequence
listed in SEQ ID NO:3 is the complementary sequence to the coding
DNA strand.
[0057] The sequence listed herein as SEQ ID NO:4 is the transgenic
fluorescence protein expression cassette having zebrafish fast
skeletal muscle specific myosin light chain (zMLC) promoter,
ZsYellow1 (a yellow fluorescent protein gene from Anthozoa, a reef
coral), and two copies of SV40 polyadenylation sequence. The
sequence listed in SEQ ID NO:4 is the complementary sequence to the
coding DNA strand.
[0058] The sequence listed herein as SEQ ID NO:5 is the transgenic
fluorescence protein expression cassette having carp ubiquitous
.beta.-actin enhancer/promoter, ZsGreen1 (a green fluorescent
protein gene from Anthozoa, a reef coral), and two copies of SV40
polyadenylation sequence. The first exon and intron of .beta.-actin
has been incorporated in the SEQ ID NO:5 to allow for increased
expression of the fluorescence gene
Chimeric Genes
[0059] The present invention encompasses chimeric genes comprising
a promoter described herein operatively linked to a heterologous
gene. Thus, a chimeric gene can comprise a promoter of a zebrafish
operatively linked to a zebrafish structural gene other than that
normally found linked to the promoter in the genome. Alternatively,
the promoter can be operatively linked to a gene that is exogenous
to a zebrafish, as exemplified by the DsRed2 and other genes
specifically exemplified herein. Furthermore, a chimeric gene can
comprise an exogenous promoter linked to any structural gene not
normally linked to that promoter in the genome of an organism.
Substitutions, Additions and Deletions
[0060] As possible variants of the above specifically exemplified
polypeptides, the polypeptide may have additional individual amino
acids or amino acid sequences inserted into the polypeptide in the
middle thereof and/or at the N-terminal and/or C-terminal ends
thereof so long as the polypeptide possesses the desired physical
and/or biological characteristics. Likewise, some of the amino
acids or amino acid sequences may be deleted from the polypeptide
so long as the polypeptide possesses the desired physical and/or
biochemical characteristics. Amino acid substitutions may also be
made in the sequences so long as the polypeptide possesses the
desired physical and biochemical characteristics. DNA coding for
these variants can be used to prepare gene constructs of the
present invention.
[0061] A nucleic acid sequence "encodes" or "codes for" a
polypeptide if it directs the expression of the polypeptide
referred to. The nucleic acid can be DNA or RNA. Unless otherwise
specified, a nucleic acid sequence that encodes a polypeptide
includes the transcribed strand, the hnRNA and the spliced RNA or
the DNA representative thereof.
Degenerate Sequences
[0062] In accordance with degeneracy of genetic code, it is
possible to substitute at least one base of the base sequence of a
gene by another kind of base without causing the amino acid
sequence of the polypeptide produced from the gene to be changed.
Hence, the DNA of the present invention may also have any base
sequence that has been changed by substitution in accordance with
degeneracy of genetic code.
DNA Modification
[0063] The DNA is readily modified by substitution, deletion or
insertion of nucleotides, thereby resulting in novel DNA sequences
encoding the polypeptide or its derivatives. These modified
sequences are used to produce mutant polypeptide and to directly
express the polypeptide. Methods for saturating a particular DNA
sequence with random mutations and also for making specific
site-directed mutations are known in the art; see e.g. Sambrook et
al., (1989).
Transgenic Fish
[0064] The disclosed constructs and methods can be used with any
type of fish that is an egg-layer. It is preferred that fish
belonging to species and varieties of fish of commercial value,
particularly commercial value within the ornamental fish industry,
be used. Such fish include but are not limited to catfish,
zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs, sharks
(family cyprinidae), angelfish, loach, koi, glassfish, catfish,
angel fish, discus, eel, tetra, goby, gourami, guppy, Xiphophorus,
hatchet fish, Molly fish, or pangasius. A more complete list of
ornamental fish species can be found in Table 5 below:
TABLE-US-00005 TABLE 5 Ornamental Fish Species Scientific Name
Common Name Steatocranus casuarius African Blockhead Apistograma
agassizi Agassizi Hyphessobrycon h axelrodi, sp Albino Black Neon
Tetra Lamprophogus brichardi Albino Bricardi Cichld Paracheirodon
innessi, sp. Albino Brilliant Neon Tetra Hemigrammus caudovitatus
Albino Buenos Aires Tetra Hemigrammus erythrozonus Albino Glow
Light Tetra Hemigrammus ocellifer Albino Head Tail Light Tetra
Pelvicachromis pulcher Albino Kribensis Cichlid Aplochelius normani
Albino Lampeye Hyphessobrycon pulchripinnis sp Albino Lemon Tetra
Paracheirodon innessi Albino Neon Tetra Macropodus opercularis spp
Albino Paradise Fish Pterophyllum scalare Albino Red Eye Angel
Epalzeorhynchos frenatus Albino Redfin Shark Hem. Rhodostomus sp.
Albino Rummy Nose Capoeta tetrazona Albino Tiger Barb Astronotus
ocellatus Albino Tiger Oscar Tanichtys albonubes sp. Albino White
Cloud Lepisosteus oculatus Alligator Gar Luciosoma spilopleura
Apollo Shark Toxotes jaculatrix Archer Fish Xiphophorus variatus
Assorted Variatus Badis badis Badis Badis Helostoma temmincki
Balloon Kissing Gourami Corydoras metae Bandit Corydoras Pangasius
sutchi Bangkok Catfish Ancistrus dolichopterus Big-Fin Bristlenose
Golden Longfin Peocilia latipinna Black Balloon Molly Cichlasoma
maculicauda Black Belt Cichlid Carrasius auratus Black Butterfly
Tail Callochromis macrops Black Eared Callochromis Leptosoma
Kitumba Black Finned Slender Cichlid Apteronotus albifrons Black
Ghost Acanthopthalmus myersi Black Kuhlii Bogrichthys hypselopterus
Black Lancer Hyphessobrycon h axelrodi Black Neon Tetra
Nematobrycon palmeri spp Black Palmeri Megalamphodus megalopterus
Black Phantom Rasbora trilineata Black Scissor Tail Rasbora Labeo
chrysopekadion Black Shark Puntius filamentosus Black Spot Barb
Rasbora agilis Black Stripe Rasbora Gymnocorymbus ternetzi Black
Tetra Astyanax fas. mexicanus Blind Cave Tetra Brachydanio kerri
Blue Danio Inpaichtys kerri Blue Emperor Tetra Trichogaster
trichopterus Blue Gourami Boehlkea fredcochui Blue King Tetra
Xiphophorus maculatus Blue Platy Melanotaenia lacustris Blue
Rainbow Poecilia reticulata Blue Ribbon Guppy Pseudotropheus zebra
Blue Zebra Melanotaenia boesemani Boesemani Rainbow Gastromyzon
punctulatus Borneo Sucker Datnoides microlepis Borneo Tiger Fish
Paracheirodon innesi Brilliant Diamond Head Neon Rasbora birttani
Brittan'S Rasbora Brachygobius doriae Bumble Bee Goby
Anomalochromis thomasi Butterfly Cichlid Notesthes robusta
Butterfly Goby Paracheirodon axelrodi Cardinal Tetra - M
Nomorhampheus liemi Celebes Halfbeak Telmatherina ladigesi Celebes
Rainbow Chaca bankanensis Chaca - Chaca Capoeta oligolepis
Checkered Barb Capoeta titteya Cherry Barb Sphaerichthys
osphromenoides Chocolate Gourami Clarias batracus Clarias - Spotted
Epiplatys annulatus Clown Killie/Rocket Botia macracantha Clown
Loach Haplochromis sp Cobalt/Ice Blue Cichlid Apistograma
cacatuoides Cockatoo Dwarf Hyphessobrycon colombianus Colombia
Tetra Phenacogrammus interruptus Congo Tetra Corydoras aeneus
Corydoras Albino Corydoras panda Corydoras Panda Corydoras paleatus
Corydoras Peppered Corydoras pigmy Corydoras Pigmy Corydoras
rabauti Corydoras Rabauti Corydoras similis Corydoras Similis
Corydoras sterbai Corydoras Sterbai Synodontis multipunctatus
Cuckoo Synodontis Polypterus senegalus Cuvier'S Bichir Synodontis
decorus Decorated Synodontis Polypterus delhezi Delhezi Bichir
Moenkhausia pitteri Diamond Tetra Hyphessobrycon amandae Ember
Tetra Nematobrycon palmeri Emperor Tetra Polypterus endlicheri
Endlicheri Bichir Aphyocharax alburnus False Flame Tetra Synodontis
eupterus Feathered Fin Synodontis Cichlasoma festae Festa'S Cichlid
Cichlasoma meeki Firemouth Cichlid Puntius pentazona Five Banded
Barb Epalzeorhynchus kalopterus Flying Fox Crossocheilus siamensis
Flying Fox Popondetta furcata Forktail Rainbow Cyphotilapia
frontosa Frontosa Cichlid Cyathopharynx furcifer Furcifer Sturisoma
fursochi Fursochi Cat Fish Aphyosemion gardneri Gardneri Killifish
Pseudomugil gertrudae Gertrudae Danio malabarinchus Giant Danio
Ambassis ranga Glass Angel Prionobrama filigera Glass Bloodfin
Hypostomus plecostomus Glass Cleaner Plecostomus Hemigrammus
rodwayi Gold Tetra Puntius sachsi Golden Barb Nannacara anomala
Golden Dwarf Cichlid Nannostomus beckfordi Golden Pencil Tetra
Pristella maxillaris Golden Pristella Melanotaenia herbrt axelrodi
Golden Rainbow Scleropages formosus Green Arowana Brachydanio rerio
Green Danio Aequidens rivulatus Green Terror Cichlid Macrognathus
circumcinctus Half Banded Spiny Eel Rasbora heteromorpha Harlequin
Rasbora Gasteropelecus sternicla Hatchet Fish Rasbora dorsiocellata
High Spot Rasbora Geophagus steindachneri Hondae Humphead
Ctenolucius hujeta Hujeta Scleropages jardini Jardini Arowana
Hemichromis paynei Jewel Cichlid Melanochromis johanni Johanni
Cichlid Julidichromis dickfeldi Juldchrmis Dickfeldi Julidichromis
ornatus Julidochromis Ornatus Julidichromis transcriptus
Julidochromis Transcriptus Geophagus jurupari Jurupari Cichlid
Tropheus IKOLA Kaisar Tropheus Hyphessobrycon loweae Kitti Tetra
Stigmatogobius sadanundio Knight Goby Cyprinus Carpio Koi
Acanthopthalmus kuhlii Kuhlii Loach Lamprologus silindericus
Lamprologus Silindericus Lamprologus leleupi Lemon Cichlid
Labidochromis caeruleus Lemon Mbuna Cichlid Hyphessobrycon
pulchripinnis Lemon Tetra Ctenopoma acutirostre Leopard Bushfish
Brachydanio frankei Leopard Danio Leptosoma malasa Leptosoma Malasa
Rasbora paviei Line Rasbora Capoeta arulius Long Fin Barb Alesthes
longipinnis Long Fin Characin Rasbora einthovenii Long-Band Rasbora
Melanotaenia maccullochi Macculloch'S Rainbow Paretropheus
menoramba Madagascar Cichlid Bedotia gaeyi Madagascar Rainbow
Haplochromis compressiceps Malawi Eye Biter Ompok sp. Malay Glass
Catfish Betta splendens Male Betta Cichlasom managuense Managuense
Cichlid Polypterus palmas Marbled Bichir Xiphophorus helleri
Millenium Swordtail Monodactylus argentus Mono Angel Cyrtocara
moorii Morrii Sawbwa resplendens Naked Micro Rasbora Hyphessobrycon
h. axelrodi sp. Negro Brilliant Black Neon Melanotaenia praecox
Neon Dwarf Rainbow Aplocheillus panchax New Golden Wonder
Synodontis ocellifer Ocellated Synodontis Colisa labiosa Orange
Thick Lipped Gourami Polypterus ornatipinnis Ornate Bichir Botia
Locahanta Pakistani Loach Puntius fasciatus Panda Barb Apistogramma
pandurini Pandurini Dwarf Macropodus opercularis Paradise Fish
Cichlasoma sp. Parrot Cichla Ocellaris Peackock Bass Cichlid
Trichogaster leeri Pearl Gourami Cichlasoma carpinte Pearl Scale
Cichlid Lamprologus calvus Pearly Lamprologus Tropheus PEMBA Pemba
River Tropheus Thayeria boelkea Penguin Tetra Chalceus
macrolepidotus Pinktail Characin Mogurnda mogurnda Purple Striped
Gudgeon Rasbora sp. Rasbora Red Fin Aphyocharax rathbuni Red Belly
Tetra Cichlasoma labiatum Red Devil Moenkhausia santaefilomenae Red
Eye Tetra Pseudotrophues sp. Red Eyed Tangarine Cichlid
Mastacembelus erythrotaenia Red Fire Eel Copadichromis borleyi Red
Kadango Rasbora pauciperforata Red Line Rasbora Colossoma
macropodum Red Pacu Megalamphodus sweglesi Red Phantom Glossolepis
incisus Red Rainbow Cichlasoma severum Red Severum Cichlid Notropis
lutrensis Red Shiner Megalamphodus roseus Red Tail Yellow Phantom
Epalzeorhynchos frenatus Redfin Shark Epalzeorhynchos bicolor
Redtail Black Shark Puntius conchonius Rosy Barb Hyphessobrycon
bentosi Rosy Tetra Puntius rhombocellatus Round Banded Clown Barb
Puntius nigrofasciatus Ruby Barb Hemigrammus bleheri Rummy Nose
Tetra Arius graeffei Salmon Catfish Hyphessobrycon serpae Serpae
Tetra Hyphessobrycon serpae sp Serpae Tetra Veiltail Osteoglossum
bichirrhosum Silver Arowana Distichodus affinis Silver Distichodus
Metynnis hypsauchen Silver Dollar Selenotoca multifasciata Silver
Scat Hasemania nanna Silver Tipped Tetra Balantiocheilos
melanopterus Silver Tricolor Shark Rasbora espei Slender Wedge
Rasbora Pseudomugil signifer Southern Blue Eye Chilodus punctatus
Spotted Headstander Rasbora maculata Spotted Pygmy Rasbora Metynnis
maculatus Spotted Silver Dollar Puntius lineatus Striped/Lined Barb
Scleropages formosus Super Red Arowana Corynopoma riseii Swordtail
Characin Cichlasoma synspilum Synspillum Cichlid Iriantherina
werneri Threadfin Rainbow Capoeta tetrazona Tiger Barb
Pseudoplatystoma fasciatum Tiger Shovelnose Catfish Tilapia
buttikoferi Tiger Zebra Tilapia Petrochromis trewavasae Trewavas'S
Petrochromis Tropheus duboisi Tropheus Duboisi Mystus micracanthus
Two Spotted Catfish Uaru amphiacanthoides Uaru - Triangle Cichlid
Sphaerichthys vallianti Valliant'S Gourami Thayeria boehlkea sp.
Veiltail Penguin Tetra Opthalmotilapia ventralis Ventralis
Haplochromis venustus Venustus Synodontis schoutedeni Vermiculated
Synodntis Tanichtys albonubes White Cloud Tanichtyhs albonubes
White Cloud Minnow Osphronemus gourami White Giant Gourami
Symphysodon aequifasciata White Smoke Aphyocharax paraguayensis
White Spot Tetra Crenicichla saxalitus White Spotted Pike Cichlid
Mastacembelus armatus White Spotted Spiny Eel Gymnocorymbus
ternetzi White Tetra Betta coccina Wine Red Betta Melanochromis
auratus Yellow Auratus Cichlid Hemmigrammopetersius caudalis Yellow
Congo Apistograma borelli Yellow Dwarf Cichlid
[0065] The more preferred fish for use with the disclosed
constructs and methods is zebrafish, Danio rerio. Zebrafish are
increasingly popular ornamental animals and would be of added
commercial value in various colors. Zebrafish embryos are easily
accessible and nearly transparent. The most preferred fish for use
with the disclosed constructs and methods is the Golden Zebrafish.
Zebrafish skin color is determined by pigment cells in their skin,
which contain pigment granules called melanosomes. The number, size
and density of the melanosomes per pigment cell influence the color
of the fish skin. Golden zebrafish have diminished number, size,
and density of melanosomes and hence have lighter skin when
compared to the wild type zebrafish. Golden zebrafish have a
mutation in slc24a5 gene, slc24a5 codes for a putative cation
exchanger localized to intracellular membrane, rendering the fish
skin lighter or less pigmented (Lamason et al., 2005).
[0066] The disclosed transgenic fish are produced by introducing a
transgenic construct into the genomes of cells of a fish,
preferably embryonic cells, and most preferably in a single cell
embryo. Where the transgenic construct is introduced into embryonic
cells, the transgenic fish is obtained by allowing the embryonic
cell or cells to develop into a fish. The disclosed transgenic
constructs can be introduced into embryonic fish cells using any
suitable technique. Many techniques for such introduction of
exogenous genetic material have been demonstrated in fish and other
animals. These include microinjection (Culp et al., (1991),
electroporation (Inoue et al., 1990; Muller et al., 1993; Murakami
et al., 1994; Muller et al., 1992; and Symonds et al., 1994),
particle gun bombardment (Zelenin et al., 1991), and the use of
liposomes (Szelei et al., 1994). The preferred method for
introduction of transgenic constructs into fish embryonic cells is
by microinjection.
[0067] Embryos or embryonic cells can generally be obtained by
collecting eggs as soon as possible after they are laid by methods
that are well known to those of ordinary experience in the
ornamental fish production field. Depending on the type of fish, it
is generally preferred that the eggs be fertilized prior to or at
the time of collection. This is preferably accomplished by placing
a male and female fish together in a tank that allows egg
collection under conditions that stimulate mating. A fertilized egg
cell prior to the first cell division is considered a one cell
embryo, and the fertilized egg cell is thus considered an embryonic
cell.
[0068] The transgene may randomly integrate into the genome of the
embryo in one or more copies (concatemers). After introduction of
the transgenic construct, the embryo is allowed to develop into a
fish. The fish that were injected as embryos are allowed to
interbreed and the offspring are screened for the presence of the
transgene. Fish harboring the transgene may be identified by any
suitable means. In the preferred case, one or more of the
transgenic constructs will have integrated into the cellular
genome, which can be probed for the presence of construct
sequences. To identify transgenic fish actually expressing the
transgene, the presence of an expression product can be assayed.
Several techniques for such identification are known and used for
transgenic animals and most can be applied to transgenic fish.
Probing of potential or actual transgenic fish for nucleic acid
sequences present in or characteristic of a transgenic construct
can be accomplished by Southern or northern blotting, polymerase
chain reaction (PCR) or other sequence-specific nucleic acid
amplification techniques.
[0069] The simplest way to confirm the presence of a fluorescent
protein expressing transgene in a given fish is by visual
inspection, as the fish in question would be brightly colored and
immediately distinguishable from non-transgenic fish. Preferred
techniques for identifying fluorescent protein expressing
transgenic zebrafish are described in the examples. The present
invention also provides a method to obtain a new population or the
progenitor of a new line of fluorescent transgenic fish exhibits
strong visible fluorescence, strong visible fluorescence means that
a person with 20/20 vision (i.e., average vision) will be able to
distinguish between the fluorescent fish in question and a
non-fluorescent fish of the same species at a distance of at least
5 feet in a lighted office, with a preferred distance of at least
10 feet in a lighted office, and a more preferred distance of at
least 15 feet in a lighted office, and an even more preferred
distance of at least 20 feet in a lighted office, with the
illumination level defined in Table 6. One can observe all
transgenic fluorescent fish from a particular population that
exhibit strong visible fluorescence under the various lighting
conditions and select the fish that exhibits the highest level of
visible fluorescence of the fluorescent protein. Selected fish with
strong visible fluorescence are monitored and selected continuously
to ensure stability of expression and maintenance of the strong
visible fluorescence trait. Thus a new line of fish exhibiting
strong visible fluorescence is created for further breeding.
[0070] The invention further encompasses progeny of a transgenic
fish containing a genomically integrated transgenic construct, as
well as transgenic fish derived from a transgenic fish egg, sperm
cell, embryo, or other cell containing a genomically integrated
transgenic construct. "Progeny," as the term is used herein, can
result from breeding two transgenic fish of the invention, or from
breeding a first transgenic fish of the invention to a second fish
that is not a transgenic fish of the invention. In the latter case,
the second fish can, for example, be a wild-type fish, a
specialized strain of fish, a mutant fish, or another transgenic
fish. The hybrid progeny of these matings have the benefits of the
transgene for fluorescence combined with the benefits derived from
these other lineages.
Fertilization from Frozen Sperm
[0071] Sperm freezing methods are well known in the art, for
example see Walker and Streisinger (1983). Frozen zebrafish sperm
may be used to fertilize eggs also as described in Walker and
Streisinger (1983), incorporated herein by references. Briefly, a
droplet of ice-cold 100% Hank's saline is placed next to zebrafish
eggs in a petri dish. Frozen sperm is thawed for a few seconds in
air then expelled into the droplet of Hank's saline and the
solution is mixed with the eggs. The mixture is incubated for about
.about.1 minute and then fish water added.
Vectors
[0072] The invention is further directed to a replicable vector
containing cDNA that codes for the polypeptide and that is capable
of expressing the polypeptide.
[0073] The present invention is also directed to a vector
comprising a replicable vector and a DNA sequence corresponding to
the above described gene inserted into said vector. The vector may
be an integrating or non-integrating vector depending on its
intended use and is conveniently a plasmid. The present invention
also encompasses the removal of the vector backbone from the
plasmid before the transgenic construct may be introduced into the
zebrafish.
Transformed Cells
[0074] The invention further relates to a transformed cell or
microorganism containing cDNA or a vector which codes for the
polypeptide or a fragment or variant thereof and that is capable of
expressing the polypeptide.
Expression Systems Using Vertebrate Cells
[0075] Interest has been great in vertebrate cells, and propagation
of vertebrate cells in culture (tissue culture) has become a
routine procedure. Examples of vertebrate host cell lines useful in
the present invention preferably include cells from any of the fish
described herein. Expression vectors for such cells ordinarily
include (if necessary) an origin of replication, a promoter located
upstream from the gene to be expressed, along with a
ribosome-binding site, RNA splice site (if intron-containing
genomic DNA is used or if an intron is necessary to optimize
expression of a cDNA), and a polyadenylation site.
[0076] In another aspect of the present invention, also included is
the commercial marketability of the transgenic fluorescent fish to
the ornamental fish industry.
EXAMPLES
[0077] The invention will now be further described with reference
to the following examples. These examples are intended to be merely
illustrative of the invention and are not intended to limit or
restrict the scope of the present invention in any way and should
not be construed as providing conditions, parameters, reagents, or
starting materials which must be utilized exclusively in order to
practice the art of the present invention.
Example 1
Design and Generation of the Construct Plasmids
[0078] The promoter of the zebrafish fast skeletal muscle myosin
light chain (zMLC2) (Ju et al., 2003) and the carp .beta.-actin
enhancer/promoter sequence (Lui et al., 1990) were cloned into
pBluescript II SK (-) and pUC18 respectively. Red fluorescent
protein gene, DsRed2; green fluorescent protein gene, ZsGreen1 and
yellow fluorescent protein gene, ZsYellow1 were amplified by PCR
from pDsRed2-N1, pZsGreen1-N1 and pZsYellow1-N1 (Clontech Inc.,
Matz. et al., 1999) respectively and cloned into pBluescript II SK
(-) zMLC2 and pUC 18-carp .beta.-actin such that the promoter was
operably linked to the fluorescent gene. Tandem SV40(A) polyA/3'UTR
sequence from pK-SV40(A)X2 plasmid were cloned 3' to the
fluorescent protein gene coding region. It is preferred to use more
than one copy of the selected polyadenylation sequence, and more
preferred to use a viral polyadenylation sequence, as this will
increase the efficiency of the fluorescent protein gene expression.
The resulting five construct vector maps are provided as FIG. 1
through FIG. 5.
Example 2
Preparation of the Construct for Delivery
[0079] The vectors pUC18-carp .beta.-actin-DsRed2 and pUC18-carp
.beta.-actin-ZsGreen1 were restriction double digested with XbaI
and AatII enzymes for three hours (FIG. 6, Step 1) and then run on
0.8% agarose gel to separate the transgenic insert cassette from
the vector backbone (FIG. 6, Step 2 and 3). Transgenic insert
cassette band (.about.3.5 kb) which contained the promoter, the
open reading frame and the 3'UTR was excised and purified using
phenol:choloroform extraction.
[0080] The transgenic vectors pBluescript II
SK(-)-zMLC-DsRed2-SV40x2, pBluescript II
SK(-)-zMLC-ZsGreen1-SV40x2, and pBluescript II
SK(-)-zMLC-ZsYellow1-SV40x2 were restriction triple digested with
XhoI, XmnI and NotI enzymes for three hours and then run on 0.8%
agarose gel to separate the transgenic insert cassette from the
vector backbone. The transgenic insert cassette band (.about.3.2
kb) which contained the promoter, the open reading frame and the
3'UTR was excised and gel purified.
Example 3
Making the Transgenic Fish
[0081] The purified transgenic insert cassette which contained the
promoter, the open reading frame and the 3'UTR was microinjected
into the zebrafish embryos (FIG. 6, Step 4).
[0082] While only one construct was injected into Yellow zebrafish
1, to increase the chances of developing a fish with strong visible
fluorescence, more than one construct was injected simultaneously
in Red zebrafish 1 and Green zebrafish 1. For the purposes of this
application, strong visible fluorescence means that a person with
20/20 vision (i.e., average vision) will be able to distinguish
between the fluorescent fish in question and a non-fluorescent fish
of the same species at a distance of at least 5 feet in a lighted
office, with a preferred distance of at least 10 feet in a lighted
office, and a more preferred distance of at least 15 feet in a
lighted office, and an even more preferred distance of at least 20
feet in a lighted office, with the illumination level defined in
Table 6.
[0083] Given the same illumination levels, distances, and observer
of average vision, another preferable quality of fish that exhibit
strong visible fluorescence are those fish that also exhibit
ubiquitous expression of the fluorescence, defined herein to mean
strong fluorescence that is not limited to a particular tissue type
or body location, with such expression preferably including fins,
eyes, stripes or spots. Typically, ubiquitous fluorescent
expression will mean that the fluorescent expression is visible
over 75% to 100% of the body of the fish (excluding fins and eyes).
The inventors have discovered that the use of a ubiquitous promoter
in combination with a tissue specific promoter (such as a muscle
promoter), particularly where such fish are prepared using at least
two expression vectors, will generally result in fish having the
desirable ubiquitous expression trait. In this more preferred
example, the fluorescent pattern exhibited by the fish would also
be free from any patches of non-expression or noticeably weak or
dull expression, with the possible exception of non-expression in
fins, eyes, and stripes or spots. Expression in the fins, eyes, and
stripes or spots is also preferred, but not required for a fish to
be considered as exhibiting ubiquitous fluorescent expression.
[0084] Examples of fish exhibiting strong visible fluorescence are
the lines which are the subject of the present invention. Color
photographs of these fish are available through World Wide Web at
glofish.com/photos.asp. Color photographs of fish that are
fluorescent, yet that do not exhibit strong visible fluorescence
are available through World Wide Web at
glofish.com/old_glofish.asp.
[0085] To obtain strong visible fluorescence, it is preferred to
use a promoter that expresses ubiquitously and co-inject this
promoter with a strong muscle promoter. It is also preferred to use
enhancing elements in the transgenic insert cassette. For example,
in the present invention, both Red zebrafish 1 and Green zebrafish
1 incorporate more than one transgenic expression cassette, with
one being a ubiquitous promoter, and the other being a strong
muscle promoter. In particular, Red zebrafish 1 incorporates the
cassettes represented by FIG. 1 and FIG. 4, and Green zebrafish 1
incorporates the cassettes represented by FIG. 2 and FIG. 5. In the
cassette that includes the ubiquitous promoter, there is also an
intron and exon, which exemplifies the type of RNA processing
element that is helpful in achieving strong visible
fluorescence.
[0086] To co-inject the embryos, multiple purified transgenic
insert cassettes can simply be loaded into the microinjection
needle simultaneously and then injected. Alternatively, in the
preferred method, the injection of constructs containing multiple
(two or more) fluorescent protein expression cassettes can be made
using common molecular biology techniques, such as DNA digestion
and ligation. In the most preferred method, a plasmid can be made
which contains several fluorescent protein expression cassettes in
tandem, and then treated in the same way as disclosed herein for a
single fluorescent protein expressing plasmid (that is, made,
isolated, purified, and linearized with the antibiotic resistance
marker gene and replication origin removed before injection). While
the present invention incorporates only the transgenic insert
cassettes shown in the Figures, it is understood that multiple
transgenic insert cassettes of any type can be simultaneously
injected into a fish embryo from any species. Once injected, the
embryos were allowed to grow into adult fish. At that point, they
were spawned to determine if their offspring carried the
fluorescence trait. The preferred method of spawning is a single
pair spawn between a zebrafish that had been injected as an embryo
and a wild-type zebrafish. The offspring of the transgenic
zebrafish were raised to maturity and the fluorescent fish selected
for further examination. In the preferred method, the offspring
should be screened for by exposure to lights of specific
wavelengths while they are still embryos. For example, for green
fluorescent protein an excitation max at 493 nm with emission max
at 505 nm, for red fluorescent protein an excitation max at 563 nm
and an emission max at 582 nm and for yellow fluorescent protein an
excitation max at 529 nm and an emission max at 539 nm was used
corresponding, for example, to ZsGreen1, DsRed2 and ZsYellow1.
[0087] The foregoing method was used to screen for the most
esthetically pleasing fish while still maintaining the ability to
efficiently breed.
Example 4
Selecting the Transgenic Fish
[0088] Any fish showing fluorescence as embryos or juveniles were
grown to maturity and examined for fluorescence as an adult to
determine which specific fluorescent fish was to be used as a
progenitor for a new line. In this endeavor, the most valuable
expression pattern is one that meets the definition of a fish
exhibiting strong visible expression as defined herein, and even
more preferred are those that also exhibit ubiquitous expression,
as this strong expression would increase both the aesthetic appeal
and commercial value of the fish. In particular, it is important to
be sure the fish exhibits strong visible fluorescence in all of the
lighting conditions described in Table 6 below.
TABLE-US-00006 TABLE 6 Common Light Levels - Indoors and Outdoors
Condition Illumination (lux) Full Daylight 10,000 Overcast Day 1000
Lighted Home >150 Lighted Office 500 Dark Indoor Room <50
Moderately Lit Room 100-150
[0089] Accordingly, to ensure that a progenitor for a new line of
fluorescent fish exhibits strong visible fluorescence, one can
observe all transgenic fluorescent fish from a particular
population that exhibit strong visible fluorescence under the
various lighting conditions noted above in Table 6, and select the
fish that exhibits the highest level of visible fluorescence of the
fluorescent protein. Selection of this fish is based on visible
observation only, as commercial appeal will be based on visual
appearance. When testing the fish in a completely dark room, it is
preferred to use an ultraviolet light to observe the level of the
fish's fluorescent expression, as the ambient light will typically
be insufficient to observe even the most strongly expressing
fluorescent fish.
[0090] It is also preferred to provide fish that exhibit a
reasonably stable color over the entire life of the fish, varying
no more than about 20% at any given age as compared to very young
fish of the same line. For example, the inventors have noted that
some fish, particularly those that are not prepared by the more
preferred methods of the present invention, tend to dramatically
lose their color brilliance over time, and can become
indistinguishable from non-transgenic fish of the same species,
even as young as one year old. Preferred transgenic fish of the
present invention can be selected for this trait by monitoring the
fish over its development cycle. It is also preferred to select
fish that are stable without regard to the ambient physical
environment of the tank (e.g., color of gravel, plants, etc.). This
can be ensured by selecting fish that do no lose their color
brilliancy over time or in response to the physical
environment.
[0091] Mendelian inheritance of the fluorescent trait is consistent
with an integration event at a single locus in the selected fish.
The progeny from the originally selected zebrafish comprising this
particular transgenic event can be used for further breeding
through traditional means with unmodified zebrafish to establish a
new line of fluorescent fish through methods that are well known to
those of ordinary skill in the production of fish, wherein the vast
majority of fluorescent fish derived from this progenitor exhibited
a materially similar fluorescence pattern and strength as the
founding fish. It is also preferred that the selected fish be
monitored for stability and consistency of expression, as any
life-cycle variance from strong visible fluorescence that is seen
in the selected fish may be passed along to the offspring.
Additionally, to facilitate consistency of expression, it is also
necessary to remove from the breeding population of this line any
fish that appear from time to time with an expression pattern which
is visibly weaker than the original founder.
[0092] The specific transgenic events embodied in these fish are
designated Red zebrafish 1, Green zebrafish 1 and Yellow zebrafish
1 respectively. Sperm from these fish may be used to fertilize
zebrafish eggs and thereby breed transgenic zebrafish that comprise
these specific transgenic integration events. Sperm from each line
is deposited at the European Collection of Cell Cultures (ECACC) as
"Red zebrafish 1" (provisional accession no. 06090403), "Green
zebrafish 1" (provisional accession no. 06090401) and "Yellow
zebrafish 1" (provisional accession no. 06090402).
Example 6
Breeding the Transgenic Fish
[0093] Once the transgenic line had been established as described
above, fish that were homozygous for the fluorescence trait were
obtained by crossing fish that were heterozygous for the
fluorescence trait, and then the progeny were screened to determine
whether they were homozygous for the fluorescence trait. The
preferred method of screening the progeny is through a test cross
with a wild-type zebrafish, where any fluorescent fish that
produces 100% fluorescent offspring would be homozygous for the
fluorescent trait. Once enough homozygous fish were found to create
a minimal breeding population, they were crossed to produce
additional homozygous progeny. Upon adulthood, these progeny were
crossed with wild-type fish to obtain progeny that were
heterozygous for the fluorescent trait. These heterozygous fish
were then sold to the commercial ornamental fish market, while the
homozygous fish population was maintained through traditional
methods to ensure a future homozygous breeding population.
Example 7
Potential Application of the Transgenic Fish
[0094] The fluorescent transgenic fish have use as ornamental fish
in the market. Stably expressing transgenic lines can be developed
by breeding a transgenic individual with a wild type fish, mutant
fish or another transgenic fish. Multiple color fluorescent fish
may be generated by the same technique as red fluorescent fish,
yellow fluorescent fish and green fluorescent fish. By recombining
different tissue specific promoters and fluorescent protein genes,
more varieties of transgenic fish of different fluorescent color
patterns will be created. By expression of two or more different
fluorescent proteins in the same tissue, an intermediate color may
be created. For example, combing expression of both red fluorescent
protein gene and yellow fluorescent protein gene under a
muscle-specific promoter, an orange fluorescent zebrafish may be
created.
[0095] The fluorescent transgenic fish should also be valuable in
the market for scientific research tools because they can be used
for embryonic studies such as tracing cell lineage and cell
migration. Cells from transgenic fish expressing green fluorescent
protein can also be used as cellular and genetic markers in cell
transplantation and nuclear transplantation experiments.
Additionally these fish can be used to mark cells in genetic mosaic
experiments and in fish cancer models.
[0096] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
513119DNAArtificialCloned expression cassette 1ggccgcaaca
gcggaatgac cacatttgta gaggttttac ttgctttaaa aaacctccca 60catctccccc
tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
120gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
taaagcattt 180ttttcactgc attctagttg tggtttgtcc aaactcatca
atgaccacat ttgtagaggt 240tttacttgct ttaaaaaacc tcccacatct
ccccctgaac ctgaaacata aaatgaatgc 300aattgttgtt gttaacttgt
ttattgcagc ttataatggt tacaaataaa gcaatagcat 360cacaaatttc
acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
420catcaatgaa ctctggatca ctagtctaca ggaacaggtg gtggcggccc
tcggtgcgct 480cgtactgctc cacgatggtg tagtcctcgt tgtgggaggt
gatgtccagc ttggcgtcca 540cgtagtagta gccgggcagc tgcacgggct
tcttggccat gtagatagac ttgaactcca 600ccaggtagtg gccgccgtcc
ttcagcttca gggccttgtg ggtctcgccc ttcagcacgc 660cgtcgcgggg
gtacaggcgc tcggtggagg cctcccagcc catggtcttc ttctgcatca
720cggggccgtc ggaggggaag ttcacgccga tgaacttcac cttgtagatg
aagcagccgt 780cctgcaggga ggagtcctgg gtcacggtcg ccacgccgcc
gtcctcgaag ttcatcacgc 840gctcccactt gaagccctcg gggaaggaca
gcttcttgta gtcggggatg tcggcggggt 900gcttcacgta caccttggag
ccgtactgga actgggggga caggatgtcc caggcgaagg 960gcagggggcc
gcccttggtc accttcagct tcacggtgtt gtggccctcg taggggcggc
1020cctcgccctc gccctcgatc tcgaactcgt ggccgttcac ggtgccctcc
atgcgcacct 1080tgaagcgcat gaactcggtg atgacgttct cggaggaggc
catgaattcg tgtgaagtct 1140aagaagatca agaagagaag tctgaagccg
cgtagtgtcc tgtacttgag gggcttatat 1200actgacgaag gccccttgct
aattatagac ccaaccttaa gtgaggtttg ggaaatagca 1260gaccgagaga
gaaagaggaa aaactatgag gtagggggaa agtgatggag ggacaccggc
1320tgggatgtac tatcatctta cagtgctcag aaatagctat tggagtcttg
gaatgggatg 1380aatctgatac tgatggcagc ggcctgcatg tctcgcgttg
tctaataagg acatggggac 1440gtaagggact gtttgagctt catacaatca
cataactcaa tttgaaatga atgagctgta 1500gccactgagt ctaaagggga
cgcaactccc tcccctaaag ttagattagc agcagatcaa 1560tactggctta
tcttgcagga ttacttgttg gatggggagg gttgcagtga tagatcctga
1620tggatttcaa tccagagatt tttgagccaa tgattatgtt gttcatattt
gttcacgccc 1680tggctgatat ataagaggac gttctggtgt cttaaccctt
atttcaaatc agccctaatc 1740gaaaataggt tttaatgctt ttcctagtat
agtgatgctg taagatttcg gttcaagtgc 1800ccccaccaca cccgtccaca
tggccatgcc catattcagc actctctctc tctctctctc 1860tctctctctc
tgccgatcga cctatgacct cacccggtca cttcagtgac tgtgaggtgc
1920catttgtggg aagctgccct gtggacaagc acgtaattac ccctttttcc
cagtttttag 1980atggtcagca cactacagcc ctctgagtgg ttgatgttgg
accctcatga agagaaattt 2040gaggcttaag acagtttttt tttgggaagg
caggcataag gaacagctgt ggtgaccaaa 2100atagctaaac aatcttgagc
gctccttgct ctctttttct tgtctaattc atcagtcact 2160gttttgcccc
ccttgagttt gttgtgcatt cccttctttc tttagactaa tttgatatcc
2220tgtgagtctg tcggcttgtt ttgctcctgc ttgttccaaa attgtttaag
attagcatta 2280ttttacagta cctagaaaaa cagataatta cagtgcttag
agtaattgag tacagcccat 2340tttgaaaatg aatatttttc tccatctctc
agtgaatata ggcaatgtat tttggtgcat 2400ttaaacaaaa cagatttatt
aaacagctat atttattaaa ataatatttt agtcacaaaa 2460ttgaaagata
aaacaattta aataaagcta aatattgcaa caaaaaaatt acaacctaca
2520aattttgaac taattttttt ccattgtttt gcttctcttg attttcccct
gtttataaat 2580ttgtatttaa tatttttcaa taacatataa atatgggtgt
agtagttttt ggaccgttat 2640cataagttat tttgttaaat aagctccaga
tttagcttca ctctcagaaa taaagggcgt 2700gagctgtcac tggggtggta
cctttccaaa tgataaaaat ttgtacctta aaggtccata 2760ataaaacctc
aagggtatat attagtacct aaaaagtaca aaagtgtttc tcttaaaatt
2820tttaggcact aatatatact tttgaggtat caatatggac cctttaagta
caaatatgta 2880ccttttgaaa aggtaccacc gcagtgacag cttgcggacc
atttatttct gagagtgtac 2940tgactaatct aatgtatatg cacaaatata
acatagcttc ctattaaaaa tattaattta 3000aaagacagat ttgggtgttt
cccagtgtag ggttgcaact ggatgggcat ccgctgcata 3060aaaaatacac
tggatgagtt ggtggctcat tcctctgtgg cgatcgatac cgtcgacct
311923656DNAArtificialCloned expression cassette 2ctagaaccat
gattacgcca agcttttaga ccttcttact tttggggatt atataagtat 60tttctcaata
aatatctcat atcttactgt ggtttaactg ctgaatctaa aattttaata
120caaaagtagt tatatttgtt gtacattgta aactataact taacttcagt
ttcagagaaa 180ctcatgtgct caaaatgtaa aaaaagtttc ctgttaaata
ttttgtaaat gtattgaaga 240caaaataaga aaaaaaaaaa tataagccac
taaatcacac tgtccttggt atcagcaaga 300gattctgaca taatcagctg
tttttgttta ttactgccat tgaaggccat gtgcattagt 360cccaagttac
acattaaaaa gtcacatgta gcttaccaac atcagtgctg ttcaagcaca
420gcctcatcta ctattcaaac tgtggcacca tctaaaatat gccagaattt
ttttatttaa 480tgaatttgac cctgaaatat gtattaatat cactcctgtg
atttttttgt aatcagctta 540caattacagg aatgcaagcc tgattcatta
caagtttcac tacactttct ctgacaacat 600cacctactga actcagacca
gctagttgct ccttaagtat acaatcatgt cactaatcct 660catttcaatg
aaaaataccc ctattgtact tggtacttgg tagataacca cagagcagta
720ttatgccatt attgtgaata caataagagg taaatgacct acagagctgc
tgctgctgtt 780gtgttagatt gtaaacacag cacaggatca aggaggtgtc
catcactatg accaatacta 840gcactttgca caggctcttt gaaaggctga
aaagagcctt attggcgtta tcacaacaaa 900atacgcaaat acggaaaaca
acgtattgaa cttcgcaaac aaaaaacagc gattttgatg 960aaaatcgctt
aggccttgct cttcaaacaa tccagcttct ccttctttca ctctcaagtt
1020gcaagaagca agtgtagcaa tgtgcacgcg acagccgggt gtgtgacgct
ggaccaatca 1080gagcgcagag ctccgaaagt ttacctttta tggctagagc
cggcatctgc cgtcatataa 1140aagagcgcgc ccagcgtctc agcctcactt
tgagctcctc cacacgcagc tagtgcggaa 1200tatcatctgc ctgtaaccca
ttctctaaag tcgacaaacc cccccaaacc taaggtgagt 1260tgatctttaa
gctttttaca ttttcagctc gcatatatca attcgaacgt ttaattagaa
1320tgtttaaata aagctagatt aaatgattag gctcagttac cggtcttttt
tttctcattt 1380acgtgcgaac tctgcttaaa ctctagttat tctttattaa
tatgtggtta tttttatata 1440tgtatgttat cataactgta ctggctatgt
caggtggtaa tgactgtaac gttacgttac 1500tcgttgtagg cacgacattg
aatgggccgg tgttgaaata agtcttcaac cccttttaac 1560ctcaaaatgt
gctctggtta acaaggattt taacagctat cagtatgact gtgcggtttt
1620aaagccgtta gtgaggcacg ttgcacactt gatggatggc cggaatggga
agttctttat 1680gcaggcagtg ctgcagcagg gtgtgaccta ctttagctaa
cgttagccgg ctaaccagca 1740ttcatctgcc ggtaacttga gtctaatatt
ctctatgtga tatcgaagtg atcaaagaca 1800cgtctgttag ctcactttaa
ccaactgtag tgaaaaatag cgcagtgtgc agcccttcaa 1860gtctttcatt
taggctgatt attcaatcat tttattaact attaacgcgt tactaaacgt
1920aaggtaacgt agtcagtttt taataactgg tgaaaagtac tggttgggtt
taaatggtga 1980cttataattg tgttggaggg ggaaaccttt ttgataaagg
ctatataatc tcaaatgaat 2040gggctgagga tggtgttcac aggtgcttta
gtgaagtccg ctcgtgaaga gtcgctgaag 2100tgactgcaga tctgtagcgc
atgcgttttg gcagacggcc gttgaaattc ggttgagtaa 2160ttgataccag
gtgaggctag aggatgtaga aattcatttg tgtagaattt agggagtggc
2220ctggcgtgat gaatgtcgaa atccgttcct ttttactgaa ccctatgtct
ctgctgagtg 2280ccacaccgcc ggcacaaagc gtctcaaacc attgcctttt
atggtaataa tgagaatgca 2340gagggacttc ctttgtctgg cacatctgag
gcgcgcattg tcacactagc acccactagc 2400ggtcagactg cagacaaaca
ggaagctgac tccacatggt cacatgctca ctgaagtgtt 2460gacttccctg
acagctgtgc actttctaaa ccggttttct cattcattta cagttcagcc
2520gggtaccgaa ttcatggcct cctccgagaa cgtcatcacc gagttcatgc
gcttcaaggt 2580gcgcatggag ggcaccgtga acggccacga gttcgagatc
gagggcgagg gcgagggccg 2640cccctacgag ggccacaaca ccgtgaagct
gaaggtgacc aagggcggcc ccctgccctt 2700cgcctgggac atcctgtccc
cccagttcca gtacggctcc aaggtgtacg tgaagcaccc 2760cgccgacatc
cccgactaca agaagctgtc cttccccgag ggcttcaagt gggagcgcgt
2820gatgaacttc gaggacggcg gcgtggcgac cgtgacccag gactcctccc
tgcaggacgg 2880ctgcttcatc tacaaggtga agttcatcgg cgtgaacttc
ccctccgacg gccccgtgat 2940gcagaagaag accatgggct gggaggcctc
caccgagcgc ctgtaccccc gcgacggcgt 3000gctgaagggc gagacccaca
aggccctgaa gctgaaggac ggcggccact acctggtgga 3060gttcaagtct
atctacatgg ccaagaagcc cgtgcagctg cccggctact actacgtgga
3120cgccaagctg gacatcacct cccacaacga ggactacacc atcgtggagc
agtacgagcg 3180caccgagggc cgccaccacc tgttcctgta gactagtgat
ccagagttca ttgatgagtt 3240tggacaaacc acaactagaa tgcagtgaaa
aaaatgcttt atttgtgaaa tttgtgatgc 3300tattgcttta tttgtaacca
ttataagctg caataaacaa gttaacaaca acaattgcat 3360tcattttatg
tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct
3420ctacaaatgt ggtcattgat gagtttggac aaaccacaac tagaatgcag
tgaaaaaaat 3480gctttatttg tgaaatttgt gatgctattg ctttatttgt
aaccattata agctgcaata 3540aacaagttaa caacaacaat tgcattcatt
ttatgtttca ggttcagggg gagatgtggg 3600aggtttttta aagcaagtaa
aacctctaca aatgtggtca ttccgctgtt gacgtc
365633151DNAArtificialCloned expression cassette 3ggccgcaaca
gcggaatgac cacatttgta gaggttttac ttgctttaaa aaacctccca 60catctccccc
tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
120gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
taaagcattt 180ttttcactgc attctagttg tggtttgtcc aaactcatca
atgaccacat ttgtagaggt 240tttacttgct ttaaaaaacc tcccacatct
ccccctgaac ctgaaacata aaatgaatgc 300aattgttgtt gttaacttgt
ttattgcagc ttataatggt tacaaataaa gcaatagcat 360cacaaatttc
acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
420catcaatgaa ctctggatca ctagtctcag ggcaaggcgg agccggaggc
gatggcgtgc 480tcggtcaggt gccacttctg gttcttggcg tcgctgcggt
cctcgcgggt cagcttgtgc 540tggatgaagt gccagtcggg catcttgcgg
ggcacggact tggccttgta cacggtgtcg 600aactggcagc gcaagcggcc
accgtccttc agcagcaggt acatgctcac gtcgcccttc 660aagatgccct
gcttgggcac ggggatgatc ttctcgcagg agggctccca gttgtcggtc
720atcttcttca tcacggggcc gtcggcgggg aagttcacgc cgtagaactt
ggactcgtgg 780tacatgcagt tctcctccac gctcacggtg atgtcggcgt
tgcagatgca cacggcgccg 840tcctcgaaca ggaaggagcg gtcccaggtg
tagccggcgg ggcaggagtt cttgaagtag 900tcgacgatgt cctgggggta
ctcggtgaac acgcggttgc cgtacatgaa ggcggcggac 960aagatgtcct
cggcgaaggg caaggggccg ccctccacca cgcacaggtt gatggcctgc
1020ttgcccttga aggggtagcc gatgccctcg ccggtgatca cgaacttgtg
gccgtccacg 1080cagccctcca tgcggtactt catggtcatc tccttggtca
ggccgtgctt ggactgggcc 1140atggtggcga ccgtcgaatt cgtgtgaagt
ctaagaagat caagaagaga agtctgaagc 1200cgcgtagtgt cctgtacttg
aggggcttat atactgacga aggccccttg ctaattatag 1260acccaacctt
aagtgaggtt tgggaaatag cagaccgaga gagaaagagg aaaaactatg
1320aggtaggggg aaagtgatgg agggacaccg gctgggatgt actatcatct
tacagtgctc 1380agaaatagct attggagtct tggaatggga tgaatctgat
actgatggca gcggcctgca 1440tgtctcgcgt tgtctaataa ggacatgggg
acgtaaggga ctgtttgagc ttcatacaat 1500cacataactc aatttgaaat
gaatgagctg tagccactga gtctaaaggg gacgcaactc 1560cctcccctaa
agttagatta gcagcagatc aatactggct tatcttgcag gattacttgt
1620tggatgggga gggttgcagt gatagatcct gatggatttc aatccagaga
tttttgagcc 1680aatgattatg ttgttcatat ttgttcacgc cctggctgat
atataagagg acgttctggt 1740gtcttaaccc ttatttcaaa tcagccctaa
tcgaaaatag gttttaatgc ttttcctagt 1800atagtgatgc tgtaagattt
cggttcaagt gcccccacca cacccgtcca catggccatg 1860cccatattca
gcactctctc tctctctctc tctctctctc tctgccgatc gacctatgac
1920ctcacccggt cacttcagtg actgtgaggt gccatttgtg ggaagctgcc
ctgtggacaa 1980gcacgtaatt accccttttt cccagttttt agatggtcag
cacactacag ccctctgagt 2040ggttgatgtt ggaccctcat gaagagaaat
ttgaggctta agacagtttt tttttgggaa 2100ggcaggcata aggaacagct
gtggtgacca aaatagctaa acaatcttga gcgctccttg 2160ctctcttttt
cttgtctaat tcatcagtca ctgttttgcc ccccttgagt ttgttgtgca
2220ttcccttctt tctttagact aatttgatat cctgtgagtc tgtcggcttg
ttttgctcct 2280gcttgttcca aaattgttta agattagcat tattttacag
tacctagaaa aacagataat 2340tacagtgctt agagtaattg agtacagccc
attttgaaaa tgaatatttt tctccatctc 2400tcagtgaata taggcaatgt
attttggtgc atttaaacaa aacagattta ttaaacagct 2460atatttatta
aaataatatt ttagtcacaa aattgaaaga taaaacaatt taaataaagc
2520taaatattgc aacaaaaaaa ttacaaccta caaattttga actaattttt
ttccattgtt 2580ttgcttctct tgattttccc ctgtttataa atttgtattt
aatatttttc aataacatat 2640aaatatgggt gtagtagttt ttggaccgtt
atcataagtt attttgttaa ataagctcca 2700gatttagctt cactctcaga
aataaagggc gtgagctgtc actggggtgg tacctttcca 2760aatgataaaa
atttgtacct taaaggtcca taataaaacc tcaagggtat atattagtac
2820ctaaaaagta caaaagtgtt tctcttaaaa tttttaggca ctaatatata
cttttgaggt 2880atcaatatgg accctttaag tacaaatatg taccttttga
aaaggtacca ccgcagtgac 2940agcttgcgga ccatttattt ctgagagtgt
actgactaat ctaatgtata tgcacaaata 3000taacatagct tcctattaaa
aatattaatt taaaagacag atttgggtgt ttcccagtgt 3060agggttgcaa
ctggatgggc atccgctgca taaaaaatac actggatgag ttggtggctc
3120attcctctgt ggcgatcgat accgtcgacc t 315143153DNAArtificialCloned
expression cassette 4ggccgcaaca gcggaatgac cacatttgta gaggttttac
ttgctttaaa aaacctccca 60catctccccc tgaacctgaa acataaaatg aatgcaattg
ttgttgttaa cttgtttatt 120gcagcttata atggttacaa ataaagcaat
agcatcacaa atttcacaaa taaagcattt 180ttttcactgc attctagttg
tggtttgtcc aaactcatca atgaccacat ttgtagaggt 240tttacttgct
ttaaaaaacc tcccacatct ccccctgaac ctgaaacata aaatgaatgc
300aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
gcaatagcat 360cacaaatttc acaaataaag catttttttc actgcattct
agttgtggtt tgtccaaact 420catcaatgaa ctctggatca ctagtgcttc
aggccagggc gctggggaag gcgatggcgt 480gctcggtcag ctgccacttc
tggttcttgg cgtcgctccg gtcctcccgc agcagcttgt 540gctggatgaa
gtgccactcg ggcatcttgc tgggcacgct cttggccttg tacacggtgt
600cgaactggca ccggtaccgg ccgccgtcct tcagcagcag gtacatgctc
acgtcgccct 660tcaggatgcc ctgcttaggc acgggcatga tcttctcgca
gctggcctcc cagttggtgg 720tcatcttctt catcacgggg ccgtcggcgg
ggaagttcac gccgttgaag atgctcttgt 780ggtagatgca gttctccttc
acgctcacgg tgatgtccac gttacagatg cacacggcgc 840cgtcctcgaa
caggaagctc cggccccagg tgtagccggc ggggcagctg ttcttgaagt
900agtccacgat gtcctggggg tactcggtga agatccggtc gccgtacttg
aagccggcgc 960tcaggatgtc ctcgctgaag ggcagggggc cgccctcgat
cacgcacagg ttgatggtct 1020gcttgccctt gaaggggtag ccgatgccct
cgccggtgat cacgaacttg tggccgttca 1080cgcagccctc catgtggtac
ttcatggtca tctcctcctt caggccgtgc ttgctgtggg 1140ccatggtggc
gaccgtcgaa ttcgtgtgaa gtctaagaag atcaagaaga gaagtctgaa
1200gccgcgtagt gtcctgtact tgaggggctt atatactgac gaaggcccct
tgctaattat 1260agacccaacc ttaagtgagg tttgggaaat agcagaccga
gagagaaaga ggaaaaacta 1320tgaggtaggg ggaaagtgat ggagggacac
cggctgggat gtactatcat cttacagtgc 1380tcagaaatag ctattggagt
cttggaatgg gatgaatctg atactgatgg cagcggcctg 1440catgtctcgc
gttgtctaat aaggacatgg ggacgtaagg gactgtttga gcttcataca
1500atcacataac tcaatttgaa atgaatgagc tgtagccact gagtctaaag
gggacgcaac 1560tccctcccct aaagttagat tagcagcaga tcaatactgg
cttatcttgc aggattactt 1620gttggatggg gagggttgca gtgatagatc
ctgatggatt tcaatccaga gatttttgag 1680ccaatgatta tgttgttcat
atttgttcac gccctggctg atatataaga ggacgttctg 1740gtgtcttaac
ccttatttca aatcagccct aatcgaaaat aggttttaat gcttttccta
1800gtatagtgat gctgtaagat ttcggttcaa gtgcccccac cacacccgtc
cacatggcca 1860tgcccatatt cagcactctc tctctctctc tctctctctc
tctctgccga tcgacctatg 1920acctcacccg gtcacttcag tgactgtgag
gtgccatttg tgggaagctg ccctgtggac 1980aagcacgtaa ttaccccttt
ttcccagttt ttagatggtc agcacactac agccctctga 2040gtggttgatg
ttggaccctc atgaagagaa atttgaggct taagacagtt tttttttggg
2100aaggcaggca taaggaacag ctgtggtgac caaaatagct aaacaatctt
gagcgctcct 2160tgctctcttt ttcttgtcta attcatcagt cactgttttg
ccccccttga gtttgttgtg 2220cattcccttc tttctttaga ctaatttgat
atcctgtgag tctgtcggct tgttttgctc 2280ctgcttgttc caaaattgtt
taagattagc attattttac agtacctaga aaaacagata 2340attacagtgc
ttagagtaat tgagtacagc ccattttgaa aatgaatatt tttctccatc
2400tctcagtgaa tataggcaat gtattttggt gcatttaaac aaaacagatt
tattaaacag 2460ctatatttat taaaataata ttttagtcac aaaattgaaa
gataaaacaa tttaaataaa 2520gctaaatatt gcaacaaaaa aattacaacc
tacaaatttt gaactaattt ttttccattg 2580ttttgcttct cttgattttc
ccctgtttat aaatttgtat ttaatatttt tcaataacat 2640ataaatatgg
gtgtagtagt ttttggaccg ttatcataag ttattttgtt aaataagctc
2700cagatttagc ttcactctca gaaataaagg gcgtgagctg tcactggggt
ggtacctttc 2760caaatgataa aaatttgtac cttaaaggtc cataataaaa
cctcaagggt atatattagt 2820acctaaaaag tacaaaagtg tttctcttaa
aatttttagg cactaatata tacttttgag 2880gtatcaatat ggacccttta
agtacaaata tgtacctttt gaaaaggtac caccgcagtg 2940acagcttgcg
gaccatttat ttctgagagt gtactgacta atctaatgta tatgcacaaa
3000tataacatag cttcctatta aaaatattaa tttaaaagac agatttgggt
gtttcccagt 3060gtagggttgc aactggatgg gcatccgctg cataaaaaat
acactggatg agttggtggc 3120tcattcctct gtggcgatcg ataccgtcga cct
315353688DNAArtificialCloned expression cassette 5ctagaaccat
gattacgcca agcttttaga ccttcttact tttggggatt atataagtat 60tttctcaata
aatatctcat atcttactgt ggtttaactg ctgaatctaa aattttaata
120caaaagtagt tatatttgtt gtacattgta aactataact taacttcagt
ttcagagaaa 180ctcatgtgct caaaatgtaa aaaaagtttc ctgttaaata
ttttgtaaat gtattgaaga 240caaaataaga aaaaaaaaaa tataagccac
taaatcacac tgtccttggt atcagcaaga 300gattctgaca taatcagctg
tttttgttta ttactgccat tgaaggccat gtgcattagt 360cccaagttac
acattaaaaa gtcacatgta gcttaccaac atcagtgctg ttcaagcaca
420gcctcatcta ctattcaaac tgtggcacca tctaaaatat gccagaattt
ttttatttaa 480tgaatttgac cctgaaatat gtattaatat cactcctgtg
atttttttgt aatcagctta 540caattacagg aatgcaagcc tgattcatta
caagtttcac tacactttct ctgacaacat 600cacctactga actcagacca
gctagttgct ccttaagtat acaatcatgt cactaatcct 660catttcaatg
aaaaataccc ctattgtact tggtacttgg tagataacca cagagcagta
720ttatgccatt attgtgaata caataagagg taaatgacct acagagctgc
tgctgctgtt 780gtgttagatt gtaaacacag cacaggatca aggaggtgtc
catcactatg accaatacta 840gcactttgca caggctcttt gaaaggctga
aaagagcctt attggcgtta tcacaacaaa 900atacgcaaat acggaaaaca
acgtattgaa cttcgcaaac aaaaaacagc gattttgatg 960aaaatcgctt
aggccttgct cttcaaacaa tccagcttct ccttctttca ctctcaagtt
1020gcaagaagca agtgtagcaa tgtgcacgcg acagccgggt gtgtgacgct
ggaccaatca 1080gagcgcagag ctccgaaagt ttacctttta tggctagagc
cggcatctgc cgtcatataa 1140aagagcgcgc ccagcgtctc agcctcactt
tgagctcctc cacacgcagc tagtgcggaa 1200tatcatctgc ctgtaaccca
ttctctaaag tcgacaaacc cccccaaacc taaggtgagt 1260tgatctttaa
gctttttaca ttttcagctc gcatatatca attcgaacgt ttaattagaa
1320tgtttaaata aagctagatt aaatgattag gctcagttac cggtcttttt
tttctcattt 1380acgtgcgaac tctgcttaaa ctctagttat tctttattaa
tatgtggtta tttttatata 1440tgtatgttat cataactgta ctggctatgt
caggtggtaa tgactgtaac gttacgttac 1500tcgttgtagg cacgacattg
aatgggccgg tgttgaaata agtcttcaac cccttttaac 1560ctcaaaatgt
gctctggtta acaaggattt taacagctat cagtatgact gtgcggtttt
1620aaagccgtta gtgaggcacg
ttgcacactt gatggatggc cggaatggga agttctttat 1680gcaggcagtg
ctgcagcagg gtgtgaccta ctttagctaa cgttagccgg ctaaccagca
1740ttcatctgcc ggtaacttga gtctaatatt ctctatgtga tatcgaagtg
atcaaagaca 1800cgtctgttag ctcactttaa ccaactgtag tgaaaaatag
cgcagtgtgc agcccttcaa 1860gtctttcatt taggctgatt attcaatcat
tttattaact attaacgcgt tactaaacgt 1920aaggtaacgt agtcagtttt
taataactgg tgaaaagtac tggttgggtt taaatggtga 1980cttataattg
tgttggaggg ggaaaccttt ttgataaagg ctatataatc tcaaatgaat
2040gggctgagga tggtgttcac aggtgcttta gtgaagtccg ctcgtgaaga
gtcgctgaag 2100tgactgcaga tctgtagcgc atgcgttttg gcagacggcc
gttgaaattc ggttgagtaa 2160ttgataccag gtgaggctag aggatgtaga
aattcatttg tgtagaattt agggagtggc 2220ctggcgtgat gaatgtcgaa
atccgttcct ttttactgaa ccctatgtct ctgctgagtg 2280ccacaccgcc
ggcacaaagc gtctcaaacc attgcctttt atggtaataa tgagaatgca
2340gagggacttc ctttgtctgg cacatctgag gcgcgcattg tcacactagc
acccactagc 2400ggtcagactg cagacaaaca ggaagctgac tccacatggt
cacatgctca ctgaagtgtt 2460gacttccctg acagctgtgc actttctaaa
ccggttttct cattcattta cagttcagcc 2520gggtaccgaa ttcgacggtc
gccaccatgg cccagtccaa gcacggcctg accaaggaga 2580tgaccatgaa
gtaccgcatg gagggctgcg tggacggcca caagttcgtg atcaccggcg
2640agggcatcgg ctaccccttc aagggcaagc aggccatcaa cctgtgcgtg
gtggagggcg 2700gccccttgcc cttcgccgag gacatcttgt ccgccgcctt
catgtacggc aaccgcgtgt 2760tcaccgagta cccccaggac atcgtcgact
acttcaagaa ctcctgcccc gccggctaca 2820cctgggaccg ctccttcctg
ttcgaggacg gcgccgtgtg catctgcaac gccgacatca 2880ccgtgagcgt
ggaggagaac tgcatgtacc acgagtccaa gttctacggc gtgaacttcc
2940ccgccgacgg ccccgtgatg aagaagatga ccgacaactg ggagccctcc
tgcgagaaga 3000tcatccccgt gcccaagcag ggcatcttga agggcgacgt
gagcatgtac ctgctgctga 3060aggacggtgg ccgcttgcgc tgccagttcg
acaccgtgta caaggccaag tccgtgcccc 3120gcaagatgcc cgactggcac
ttcatccagc acaagctgac ccgcgaggac cgcagcgacg 3180ccaagaacca
gaagtggcac ctgaccgagc acgccatcgc ctccggctcc gccttgccct
3240gagactagtg atccagagtt cattgatgag tttggacaaa ccacaactag
aatgcagtga 3300aaaaaatgct ttatttgtga aatttgtgat gctattgctt
tatttgtaac cattataagc 3360tgcaataaac aagttaacaa caacaattgc
attcatttta tgtttcaggt tcagggggag 3420atgtgggagg ttttttaaag
caagtaaaac ctctacaaat gtggtcattg atgagtttgg 3480acaaaccaca
actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
3540tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
attgcattca 3600ttttatgttt caggttcagg gggagatgtg ggaggttttt
taaagcaagt aaaacctcta 3660caaatgtggt cattccgctg ttgacgtc 3688
* * * * *