U.S. patent application number 10/771949 was filed with the patent office on 2004-08-19 for genetic manipulation of spermatogonia.
Invention is credited to DiTullio, Paul A., Ebert, Karl M..
Application Number | 20040163141 10/771949 |
Document ID | / |
Family ID | 22113398 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040163141 |
Kind Code |
A1 |
DiTullio, Paul A. ; et
al. |
August 19, 2004 |
Genetic manipulation of spermatogonia
Abstract
The invention features a method of delivering DNA to a
spermatogonium by infusing DNA in situ into a testicle of a
non-human animal and administering a condition or substance to the
testicle to increase uptake of DNA by the spermatogonium
Inventors: |
DiTullio, Paul A.;
(Northboro, MA) ; Ebert, Karl M.; (Millbury,
MA) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
22113398 |
Appl. No.: |
10/771949 |
Filed: |
February 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10771949 |
Feb 3, 2004 |
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09247246 |
Feb 9, 1999 |
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6686199 |
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60073386 |
Feb 9, 1998 |
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Current U.S.
Class: |
800/21 ;
435/461 |
Current CPC
Class: |
A01K 2227/101 20130101;
A01K 2217/05 20130101; A01K 2227/107 20130101; A01K 2227/108
20130101; A01K 2227/103 20130101; C12N 2799/021 20130101; A01K
67/027 20130101; A01K 2227/105 20130101; A01K 2227/30 20130101;
C12N 15/87 20130101; A01K 2227/102 20130101 |
Class at
Publication: |
800/021 ;
435/461 |
International
Class: |
C12N 015/87 |
Claims
What is claimed is:
1. A method of delivering a DNA to a spermatogonium, comprising
infusing in situ DNA into a testicle of a non-human animal and
administering a condition or substance to said testicle to increase
uptake of said DNA by said spermatogonium.
2. The method of claim 1, wherein said condition is passage of an
electrical current through the testicle.
3. The method of claim 2, wherein said electrical current is
applied to said testicle using a defibrillator.
4. The method of claim 2, wherein said electrical current is
applied to said testicle using an elect roej aculator.
5. The method of claim 1, wherein said substance is a lipid or a
phospholipid.
6. The method of claim 1, wherein said DNA is infused in a volume
of at least 0.1 ml per testicle.
7. The method of claim 1, wherein the epididymis is surgically
exposed at the head and the DNA is delivered to the testes via a
retrograde flush through the rete testes into the seminiferous
tubules.
8. The method of claim 1, wherein said DNA is introduced into said
spermatogonium by viral infection.
9. The method of claim 1, wherein said non-human animal is selected
from the group consisting of a sheep, goat, pig, cow, chicken,
rabbit, rat, mouse, and guinea pig.
10. The method of claim 9, wherein said non-human animal is a
pig.
11. The method of claim 1, wherein said animal is prepubetal.
12. The method of claim 1, wherein said DNA comprises a sequence
encoding a selectable marker.
13. The method of claim 12, wherein said selectable marker is
selected from the group consisting of antibiotic resistance gene, a
cell surface antigen, or thymidine kinase.
14. The method of claim 1, wherein DNA is administered to said
testicle before the time at which sperm production is detected.
15. The method of claim 9, wherein the age of said pig is at least
30 days.
16. The method of claim 15, wherein the age of said pig is not
greater than 100 days.
17. The method of claim 1, wherein said DNA is naked.
18. A method of making a non-human transgenic animal comprising
infusing in situ DNA into a testicle of a prepubetal non-human
animal, harvesting sperm cells from said animal, contacting an ovum
with said sperm cells under conditions suitable for fertilization
to produce said non-human transgenic animal.
19. The method of claim 18, wherein said prepubetal non-human
animal is a pig.
20. The method of claim 18, wherein said pig is at least 30 days
but not greater than 100 days of age.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from provisional patent
application serial No. 60/073,386, filed on Feb. 9, 1998, the
contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The invention relates to the production of transgenic
animals.
[0003] The field of transgenics has grown rapidly since the initial
experiments describing the introduction of foreign DNA into the
developing zygote or embryo (Brinster, R. L. et al., Proc. Nati.
Acad. Sci. USA 82:4438-4442 (1985), Wagner et al., U.S. Pat. No.
4,873,191 (1989)). Transgenic technology has been applied to both
laboratory and domestic species for the study of human diseases
(see, e.g., Synder, B. W., et al., Mol. Reprod. and Develop.
40:419-428 (1995)), production of pharmaceuticals in milk (see, for
review article, Ebert, K. M. and J. P. Seigrath, "Changes in
Domestic Livestock through Genetic Engineering" in Applications in
Mammalian Development, Cold Spring Harbor Laboratory Press, 1991.),
develop improved agricultural stock (see, e.g., Ebert, K. M. et
al., Animal Biotechnology 1:145-159 (1990)) and xenotransplantation
(see, e.g., Osman, N., et al., Proc. Natl. Acad. Sci USA
94:14677-14682 (1997)). However, the technique is limiting in that
it only allows for the addition of genetic material to the
developing embryo and not the deletion or modification of the
endogenous genes. In addition, the microinjection of DNA into the
nucleus is an inefficient process resulting in only 1-2% transgenic
offspring from embryos injected and frequently producing mosaic
animals which do not have the transgene in all cells.
SUMMARY OF THE INVENTION
[0004] The invention features a method of delivering DNA to a
spermatogonium by infusing DNA in situ into a testicle of a
non-human animal and administering a condition or substance to the
testicle to increase uptake of DNA by the spermatogonium. By "in
situ" is meant in the original location in the body of the animal.
Surgical exposure of the testis is not required. For example, a
needle is inserted directly into testicular tissue and DNA
delivered using a syringe. In some cases, the epididymis is
surgically exposed at the head, and the DNA is delivered to the
testes via a retrograde flush through the rete testes into the
seminiferous tubules.
[0005] By "spermatogonium" is meant an unspecialized diploid germ
cell which can undergo mitosis and meiosis to give rise to a sperm
cell. For example, a spermatogonium is a primordial germ cell which
can differentiate into a sperm cell. Preferably, the spermatogonium
is located on the walls of the basal membrane of the seminiferous
tubule. To increase uptake of DNA by a spermatogonium, the testicle
is exposed to a condition such as passage of an electrical current
through the testicle. The electrical current is applied using a
defibrillator or electroejaculator. Application of an electrical
current is a means to electroporate the DNA into the target cells,
i.e., spermatogonia. DNA uptake may also be enhanced by
administering a substance such as a lipid or phospholipid. The
condition or substance is administered to testicle either
simultaneously with the infusion of DNA or after the DNA has been
infused. DNA is administered as naked DNA or by viral infection,
e.g., packaged into a viral vector such as adenovirus,
adeno-associated virus. By "naked" is meant free from any delivery
vehicle that facilitates entry into the cell. For example, a naked
DNA preparation is free of viral proteins or particles,
DEAE-dextran, phospholipids, lipids, or calcium phosphate. The DNA
contains a sequence encoding a selectable marker such as DNA
encoding an antibiotic resistance gene, a cell surface antigen, or
thymidine kinase. Following administration of the DNA, transformed
or transfected cells are selected by administering an antibiotic,
antibody-toxin complex, or chemical agent either systemically or
locally to the testes to kill cells which do not express the DNA or
to identify cells which express the DNA. The terms "transform" and
"transfect" are used interchangeably throughout the specification;
these terms refer to means of transferring or delivering DNA to a
cell. A "transfected" or "transformed" cell is one that contains
the DNA sought to be delivered to it. The DNA may also contain a
second promoter which directs expression of an apoptotic gene to
selectively kill germ cells which have not undergone homologous
recombination with the administered DNA. The DNA is administered in
a volume of solution sufficient to infuse the entire testicle,
e.g., 0.1 ml per testicle/per treatment for a newborn animal and up
to 5 ml per testicle/per treatment for an adult animal. Repeated
treatments may be carried out if desired, e.g., to increase DNA
uptake.
[0006] Preferably, the non-human animal is a sheep, goat, pig, cow,
chicken, rabbit, rat, mouse, or guinea pig. More preferably, the
animal is prepubetal, e.g., at an age at which the testicle has not
yet begun to produce sperm. For example, the preferred age of a pig
is at least 30 days but not greater than 100 days. At this age, the
number of target cells, i.e., spermatogonia, is relatively low. An
advantage of this approach is that destruction of spermatogenic
cells prior to administration of DNA is not required.
[0007] Also within the invention is a method of making a non-human
transgenic animal comprising by infusing DNA in situ into a
testicle of a prepubetal non-human animal, harvesting sperm cells
from the animal, contacting an ovum with said the cells under
conditions suitable for fertilization, and producing a non-human
transgenic animal. By "transgenic non-human animal" is meant an
animal that has gained (or lost) a DNA sequence from the
introduction of an exogenous DNA sequence, i.e., transgene, into
its own cells, or into an ancestor's germ line. Such animals are
produced by natural breeding, artificial insemination, or in vitro
sperm injection into ova. By the term "transgene" is meant any
exogenous DNA sequence which is introduced into both the somatic
and germ cells or only some of the somatic cells of a mammal. The
transgene may or may not be an integral part of a chromosome. If
the transgene is integrated into a chromosome, it may or may not be
located at the same site as its corresponding endogenous gene
sequence.
[0008] The transgene is preferably driven by a tissue specific
promoter. For example, for protein expression in mammary glands,
the human lactoferrin promoter is operably linked to DNA encoding a
desired protein or polypeptide. Other promoters which direct
preferential expression of a polypeptide in mammary gland tissue
include the casein promoters (e.g., goat beta casein promoter and
alpha Si casein promoter) and whey protein promoters (e.g., whey
acid protein promoter, beta lactoglobulin promoter, and alpha
lactalbumin promoter). The promoter is inducible or
constitutive.
[0009] Organs from transgenic animals produced according to the
invention are useful for xenotransplantation. For example, the
transgenic non-human animal organs of which are suitable for
transplantation into human recipients expresses human CD59 and/or
lacks expression of porcine CD59 (a CD59 "knockout" pig) or the
animal expresses human H-transferase and/or lacks porcine
Gal(.alpha.,3) galactosyl transferase.
[0010] The methods of the invention solve many ongoing problems in
DNA delivery for the generation of transgenic animals. For example,
an adenovirus genome can only carry a transgene of limited size.
The use of sperm does not limit the transgene size. For example,
DNA as small as a few base pairs (bp) and as large as 100-200
kilobases (kb) are delivered using the methods of the invention.
Typically, approximately 5 kb, 10 kb, 20 kb or 25 kb are delivered
to target cells. However, up to 400 kb may be transferred to cells.
For example, more than one vector or DNA fragment is transfected
simultaneously to allow for the production of multimeric proteins
(i.e., immunoglobulins, FAb fragments, fibrinogen, and collagen) or
expression of more than one protein coding sequence.
[0011] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1A is a diagram of a cross-section of a seminiferous
tubule in a mammalian testes.
[0013] FIG. 1B is a diagram showing the organization of different
cell types within the seminiferous tubules and the respective cell
types.
[0014] FIG. 2 is a diagram showing the stages of
spermatogenesis.
DETAILED DESCRIPTION
[0015] Transgenic animals made using the methods described herein
are used for xenotranplantation, pharmaceutical production, protein
production, and the study of human diseases.
[0016] The efficiency and reliability of the production of the
transgenic animals is enhanced by the claimed methods which involve
direct transfection of the stem/progenitor cells of the male testes
with DNA. Following DNA delivery, semen is collected, tested for
the presence of the transgene, and any animals with the transgene
present in their sperm are bred to females to produce transgenic
offspring. This invention has several advantages over the
conventional microinjection approach in that a single experiment
could produce multiple transgenic lines, no mosaic animals would be
generated, the anesthetic and surgical procedures are less complex
and invasive, and the need for multiple surgeries is
eliminated.
[0017] For example, the method is carried out as follows. A male
animal is anesthetized with a suitable anesthetic to achieve a
30-60 minute time period to complete the procedure (suitable
anesthetics are known in the art, e.g., goats, valium/ketamine;
rabbits, acepromazine/rompun; pigs, telazol/rompun). The optimum
age for the male is prepubetal to reduce the number of target cells
(e.g., stem/progenitor cells) to be transfected. However, the
procedure is also useful for DNA delivery to spermatogonia of adult
or mature animals. Once the animal has been anesthetized, DNA is
introduced into the testes using a small gauge needle. The volume
of DNA and concentration varies depending on the size of the testes
and the efficiency of transfection. The transfection of the DNA is
achieved using a variety of standard techniques and DNA
formulations. For example, the DNA is infused in complex with
lipids, other compounds known to enhance transfection, or
uncomplexed (i.e., naked). If DNA is infused in sterile distilled
water, it may be electroporated into the cells in vivo by passing a
current across the testes using an electroejaculator, heart
defibrillator, or any suitable source of electrical current. After
the animal has recovered, semen is collected and analyzed for the
presence of the transgene. Once the transgene has been detected,
the male will be bred to females to produce transgenic offspring.
The time between infusion of the DNA and breeding varies depending
on such factors as the age of the animal at infusion, age of sexual
maturity, and the time required for differentiation from
stem/progenitor cell to sperm cell.
Direct Delivery of DNA to Sperm Cells/Spermatagonia
[0018] The invention described herein is a method for producing a
non-human transgenic animal by genetically manipulating the stem
cells/spermatogonia of the male testes in vivo. The testes are
composed of a series of tightly coiled tubes, termed seminiferous
tubules. Testes are derived from two cell types; cells of the
mesonephros region form the structural cells and the primordial
germ cells give rise to the spermatogonia (FIGS. 1A and B). The
seminiferous tubules contain the stem cells (spermatogonia) which
when properly stimulated will undergo mitosis and meiosis to form
mature sperm allowing reproduction (FIG. 2). Spermatogenesis begins
in puberty and continues throughout the adult life. Therefore, the
spermatogonia of the male represent the only truly regenerating
stem cell population in mammals, and the introduction of DNA into a
spermatogonia results in a continuous production of transgenic
sperm. All transgenic animals produced by this technique carry the
transgene in their germline because the DNA is integrated in the
sperm before fertilization. The DNA delivery method described
herein is used to transfect the spermatogonia in vivo. The approach
is also useful to transfect cells in vitro. For example,
spermatagonia-derived cells are cultured and transfected in vitro
to be used as a sources of totipotent nuclei for cloning.
[0019] DNA is delivered to a stem cell spermatogonia via the
seminiferous tubules using methods known in the art. For example, a
small gauge needle is inserted into testicular tissue, and DNA is
infused using a small syringe. Alternatively, the testicle is
exteriorized, a tube surgically attached to the head of the
epididymis, and DNA infused through the tube.
[0020] The methods of the invention are not limited by any
particular formulation of DNA. For example, DNA is naked or
complexed with lipids, phospholipids, or other suitable compounds
to facilitate DNA uptake by cells (i.e., DEAE-dextran). The DNA
contains one or more sequences which target expression to a
specific tissue or are non-tissue specific, aid in transgene
integration into a chromosome (e.g., viral long terminal repeat
(LTR) sequences), or cause the transgene to replicate episomally.
Such DNA constructs are made using methods well known in the art of
molecular biology. If desired, the DNA is complexed with a lipid or
other suitable compounds to facilitate DNA uptake (i.e.
DEAE-dextran). A virus capable of infecting mammalian cells is also
a useful delivery vehicle.
[0021] Transfection efficiency is increased by passing an
electrical current across the testicle with a device such as a
standard human heart defibrillator or an electroejaculator such as
that described in U.S. Pat. No. 4,564,024. The efficiency of DNA
delivery is assayed by collection of semen from the transformed
testicle. DNA is isolated from the testicle (e.g., from semen)
using standard methods, and is assayed for presence of the
transgene by methods known in the art, e.g., the polymerase chain
reaction (PCR) with transgene specific primers. If the semen tests
positive for the desired transgene, transgenic animals are produced
by breeding the male to females in estrous and allowing the animals
to deliver. The progeny are tested for the presence of the
transgene by PCR or southern blot analysis.
[0022] The overall efficiency of the procedure is dependent on the
method of gene delivery but can be increased in several ways such
as 1) subjecting the testicle to multiple rounds of transfection
with the DNA vector, or 2) adding a selectable marker, e.g., an
antibiotic resistance gene, to the DNA vector and infusing the
testicle with the antibiotic or drug following transfection to
select for the transgenic cells.
EXAMPLE 1
Timing of Testes Transfection
[0023] Experiments were carried out to determine the optimal age of
animal at which in situ DNA delivery to the testes is most
efficient. As a male animal matures, the testes increase in size.
The increase in size corresponds to an increase in the number of
target cells and primordial germ cells/spermatogonia. As a result,
partial sterilization may be required prior to DNA delivery to
mature animals.
[0024] According to the invention, the testes are transfected at an
age when the seminiferous tubules are open to allow the DNA access
to the spermatogonia but before the testes begin to actively
produce sperm by meiosis. Since different species mature at
different rates, this age varies among species. The optimal age for
any given species is determined using known methods, e.g.,
monitoring a maturing animal for the onset of sperm production or
histologically examining the testes from newborn to adult. Once the
preferred age for DNA delivery is determined for a given species,
this process need not be repeated for each individual to be
treated.
[0025] The optimal time for transfecting the testicles of a
Yorkshire boar was determined. Development of the testes from birth
to over one year of age was monitored by collecting semen and
histologically examining fixed tissue sections of testes. A gilt in
heat was used to stimulate the boar. Semen was first detected
between 4 and 5 months of age. Analysis of the semen revealed that
the sperm count was low compared to a fully mature boar. These data
indicated that the testes were still undergoing mitosis to increase
cell number as well as meiosis to produce sperm.
[0026] Gross anatomical examination of the testicles indicated that
the testicles increased in size from 5-7 cm to over 14 cm between 5
and 18 months of age. Tissue sections from a newborn (10-30 days
old) to an adult (over 12 months) were histologically examined. In
the newborn, the testicle was 1-2 cm in length, and the
seminiferous tubules were only populated by germ cells. Attempts to
infuse the testicles with tracking dye indicated that the
seminiferous tubules were not completely open to allow flow of dye
throughout the testicle. Histological examination of testicles
harvested from boars between 80-100 days of ages indicated that the
seminiferous tubules are open and showed an increase in the number
of cells (e.g., spermatogonia) lining the tubule. At this age, the
seminiferous tubules allowed transport of the tracking dye. These
data indicate that DNA can successfully be delivered throughout the
testicle via the seminiferous tubules after the age at which the
tubules have matured to be open.
[0027] In an adult boar, histological examination of tissue
sections revealed a hierarchy of cells lining the tubules with
motile sperm being produced in the lumen. The seminiferous tubules
of the adult animal also successfully carried tracking dye.
However, the larger size of the testicle at the adult stage may
reduce efficiency of DNA delivery and transfection. These data
indicate that the preferred age for DNA delivery and transfection
of the testicle of a Yorkshire boar is between 30 and 100 days of
age.
EXAMPLE 2
Transfection of Testes
[0028] The method described below is applicable to all species
which reproduce through the production of sperm, for example but
not limited to mice, rats, rabbits, pigs, goats, sheep and cows.
DNA is delivered to the area surrounding the spermatogonia by
utilizing the seminiferous tubules as conduits to transport the
genetic material throughout the testes.
[0029] To increase overall efficiency of DNA delivery, prepubetal
animals are used (thereby reducing the number of target cells in
the testes to be transfected). Use of non-viral DNA is preferable
because this approach removes any size limitation on the vector.
The protocol described herein is useful for any DNA formulation
(i.e., for either naked DNA or DNA in complex with compounds such
as lipids or DEAE-dextran). Alternatively, a virus suitable to
infect target cells of the testes (stem cells) is also a useful
vehicle to deliver DNA to the target cells.
[0030] The resulting phenotype of the animal produced by a
transformed sperm is dependent on the type of the DNA introduced
and is not dependent on the method being described. Introduction of
a vector or transgene containing mammary gland-specific regulatory
elements directs expression of the desired protein in the animal's
milk whereas a liver-specific promoter would target the blood. For
example, the following tissue-specific promoters are used to direct
preferential expression of DNA in mammary gland tissue: goat beta
casein promoter (Ebert et al., 1994, Bio/Technology 9:669-702),
alpha S1 casein promoter (Meade et al., 1990, Bio/Technology
8:443-446), whey acid protein promoter (Ebert et al., 1991,
Bio/Technology 9:835-838), beta lactoglobulin promoter (Wright et
al., Bio/Technology 9:830-834). The transcription of a transgene is
preferably under the control of a promoter sequence different from
the promoter sequence controlling the transcription of the
endogenous coding sequence, e.g., a heterologous promoter. A
promoter includes a cis-acting DNA sequence which is capable of
directing the transcription of a gene in the appropriate tissue
environment and in response to physiological regulators. Regardless
of the vector or virus system being used, this method provides an
efficient method to genetically manipulate the stem cells of the
testes to produce transgenic animals.
[0031] The methods of the invention are useful to delivery DNA to
large mammals, e.g., pigs, goats, cows, and sheep. A prepubetal
male pig between 1 and 2 months of age is anesthetized with a
mixture of telazol and rompun. Once the pig is under anesthesia, it
is placed on a surgical table, and the area around the testes is
cleaned and sterilized with alternating washes of betadine and 70%
alcohol. The DNA, which has been drawn up into a 3 cc syringe with
a 20 gauge needle, is infused into the testes by inserting the
needle through the skin and into the organ. As the solution is
infused, the needle is slowly pulled out to ensure that the entire
testicle is covered. Once the DNA is infused in to the testicle, an
electrical current is passed across the testicle with a human heart
defibrillator that has been modified to accommodate the size of the
organ. Following the transfection, the pig is allowed to recover
from anesthesia and housed until it reaches maturity at
approximately 5 months of age. Semen is collected from the pig for
analysis. The sperm are recovered from the semen by centrifugation,
washed with phosphate buffered saline, and DNA isolated by
digestion with protease followed by phenol extraction and ethanol
precipitation. The DNA isolated from the sperm is analyzed for the
presence of the transgene by the PCR with primers specific for the
transgene.
[0032] Successful "knockout" of DNA sequesces is also evaluated
using PCR or other techniques know in the art:
[0033] If the sperm DNA tests positive for the gene of interest,
transgenic animals are produced by breeding the pig to sows in
heat, using the sperm for artificial insemination, or doing in
vitro fertilization with the sperm. The recipient females are
allowed to farrow and the progeny tested for the presence of the
transgene (or knockout) with DNA isolated from ear tissue or blood
by the polymerase chain reaction.
[0034] Other embodiments are within the following claims.
* * * * *