U.S. patent application number 11/676790 was filed with the patent office on 2007-06-28 for transgenic non-human mammal with an oncogenic mutant of the c-raf-1 gene.
Invention is credited to Ulf R. RAPP.
Application Number | 20070150974 11/676790 |
Document ID | / |
Family ID | 7851363 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070150974 |
Kind Code |
A1 |
RAPP; Ulf R. |
June 28, 2007 |
TRANSGENIC NON-HUMAN MAMMAL WITH AN ONCOGENIC MUTANT OF THE c-Raf-1
GENE
Abstract
The invention relates to a transgenic non-human mammal whose
cells express a constitutively active oncogenic mutant of the
kinase-domain of the Raf-1 gene or a protein coded by a
corresponding normal allele or derivative of the A, B, or c-Raf-1
gene.
Inventors: |
RAPP; Ulf R.; (Wurzburg,
DE) |
Correspondence
Address: |
McGLEW AND TUTTLE, P.C.
SCARBOROUGH STATION
SCARBOROUGH
NY
10510-0827
US
|
Family ID: |
7851363 |
Appl. No.: |
11/676790 |
Filed: |
February 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11381024 |
May 1, 2006 |
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11676790 |
Feb 20, 2007 |
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10371138 |
Feb 20, 2003 |
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11381024 |
May 1, 2006 |
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09555279 |
Jul 17, 2000 |
6566581 |
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10371138 |
Feb 20, 2003 |
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Current U.S.
Class: |
800/18 |
Current CPC
Class: |
C12N 9/1205 20130101;
C12N 15/8509 20130101; C07K 14/82 20130101; A01K 67/0278 20130101;
C12N 2830/008 20130101; A01K 67/0275 20130101; A01K 2217/072
20130101; A01K 2227/105 20130101; A01K 2267/0331 20130101; A01K
2217/05 20130101 |
Class at
Publication: |
800/018 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 28, 1997 |
DE |
197 54 774.5 |
Claims
1. A transgenic mouse whose cells express a constitutively active
oncogenic mutant of the kinase domain of the c-Raf-1 gene or a
protein coded by a corresponding normal allele of the Raf gene or a
derivative of the A Raf gene, the B Raf gene or the c-Raf-1
gene.
2. The transgenic mouse according to claim 1, wherein the
expression takes place in lung cells.
3. A transgenic mouse, wherein the mouse contains a foreign DNA
having a constitutively active oncogenic mutant of the
kinase-domain of the c-Raf-1 gene or with a corresponding normal
allele of the Raf gene or a derivative of the A Raf gene, the B Raf
gene or the c-Raf-1 gene.
4. The transgenic mouse according to claim 3, wherein the foreign
DNA in addition contains a promoter for the surfactant protein C
and wherein this promoter is arranged in the foreign DNA with the
proviso that by the promoter the transcription of the mutant of the
kinase-domain of the c-Raf-1 gene or of a corresponding normal
allele of the Raf gene or a derivative of the A Raf gene, the B Raf
gene or the c-Raf-1 gene is controlled.
5. The transgenic mouse according to claim 3, wherein the foreign
DNA in addition contains DNA of the SV40 virus.
6. A transgenic mouse, which is obtainable by the following steps:
a) integration of the cDNA sequence of a constitutively active
oncogenic mutant of the kinase domain of the c-Raf-1 gene or of a
corresponding normal allele of the Raf gene or a derivative of the
A Raf gene, the B Raf gene or the c-Raf-1 gene in an expression
vector to provide an expression vector carrying a foreign DNA, b)
insertion of the transgenic vector obtained in step a) in pronuclei
of fertilized oocytes from a mouse, c) implantation of the oocytes
obtained in step b) in brood animals of the same species as the
donor species of the oocytes and delivery of descendant animals
from the oocytes, d) genotypization and selection of the descendant
animals obtained in step c) with the proviso that cells of the
selected mice express a constitutively active oncogenic mutant of
the kinase domain of the c-Raf-1 gene or a protein coded by a
corresponding normal allele of the Raf gene or a derivative of the
A Raf gene, the B Raf gene or the c-Raf-1 gene.
7. The transgenic mouse according to claim 6, wherein the
expression vector used in step a) contains a promoter for the
surfactant protein C and wherein this promoter is arranged in the
foreign DNA with the proviso that by the promoter the transcription
of the mutant of the kinase-domain of the c-Raf-1 gene or of a
corresponding normal allele of the Raf gene or a derivative of the
A, B or c-Raf-1 gene is controlled.
8. The transgenic mouse according to claim 3, wherein the
constitutively active oncogenic mutant of the c-Raf-1 gene
comprises a sequence according to FIG. 1 or a sequence
.DELTA.Raf(26-302) derived therefrom.
9. A method for producing a transgenic mouse, including the
following steps: a) integration of the cDNA sequence of a
constitutively active oncogenic mutant of the kinase domain of the
c-Raf-1 gene or of a corresponding normal allele of the Raf gene or
a derivative of the A Raf gene, the B Raf gene or the c-Raf-1 gene
in an expression vector to provide an expression vector carrying a
foreign DNA, b) insertion of the transgenic vector obtained in step
a) in pronuclei of fertilized oocytes from a mouse, c) implantation
of the oocytes obtained in step b) in brood animals of the same
species as the donor species of the oocytes and delivery of
descendant animals from the oocytes, d) genotypization and
selection of the descendant animals obtained in step c) with the
proviso that cells of the selected mice express a constitutively
active oncogenic mutant of the kinase domain of the c-Raf-1 gene or
a protein coded by a corresponding normal allele of the Raf gene or
a derivative of the A Raf gene, the B Raf gene or the c-Raf-1
gene.
10. The method according to claim 9, wherein the expression vector
used in step a) contains a promoter for the surfactant protein C
and wherein this promoter is arranged in the foreign DNA with the
proviso that by the promoter the transcription of the mutant of the
kinase-domain of the c-Raf-1 gene or of a corresponding normal
allele of the Raf gene or a derivative of the A; B or c-Raf-1 gene
is controlled.
11. The utilization of a mouse according to claim 1 for the
pre-clinic examination of the effectiveness of substances intended
against lung carcinomas and/or of therapeutical methods intended
against lung carcinomas.
12. The utilization according to claim 11 for the preclinic
examination of the effectiveness of substances inhibiting
Rat-kinase,
13. A lung cell tissue from a transgenic mouse according claim 1,
which cell tissue has a higher probability of forming lung
tumors.
14. A method for producing cell tissue from a transgenic mouse,
including the following steps: a) integration of the cDNA sequence
of a constitutively active oncogenic mutant of the kinase domain of
the c-Raf-1 gene or of a corresponding normal allele of the Raf
gene or a derivative of the A Raf gene, the B Raf gene or the
c-Raf-1 gene in an expression vector to provide an expression
vector carrying a foreign DNA, b) insertion of the transgenic
vector obtained in step a) in pronuclei of fertilized oocytes from
a mouse, c) implantation of the oocytes obtained in step b) in
brood animals of the same species as the donor species of the
oocytes and delivery of descendant animals from the oocytes, d)
genotypization and selection of the descendant animals obtained in
step c) with the proviso that cells of the selected mice express a
constitutively active oncogenic mutant of the kinase domain of the
c-Raf-1 gene or a protein coded by a corresponding normal allele of
the Raf gene or a derivative of the A Raf gene, the B Raf gene or
the c-Raf-1 gene, e) removal of cell tissue from the mouse.
15. The method according to claim 14, wherein the expression vector
used in step a) contains a promoter for the surfactant protein C
and wherein this promoter is arranged in the foreign DNA with the
proviso that by the promoter the transcription of the mutant of the
kinase-domain of the c-Raf-1 gene or of a corresponding normal
allele of the Raf gene or a derivative of the A Raf gene, the B Raf
gene or the c-Raf-1 gene is controlled.
16. The utilization of a cell tissue according to claim 13 for the
pre-clinic examination of the effectiveness of substances intended
against lung carcinomas and/or of therapeutical methods intended
against lung carcinomas.
17. The utilization according to claim 16 for the preclinic
examination of the effectiveness of substances inhibiting
Raf-kinase.
18. The transgenic mouse according to claim 4, wherein the promoter
for the surfactant protein C is for the human surfactant protein
C.
19. The transgenic mouse according to claim 6, wherein insertion of
the transgenic vector obtained in step occurs after
linearization.
20. The transgenic mouse according to claim 6, wherein the promoter
for the surfactant protein C is for the human surfactant protein
C.
21. The method according to claim 9, wherein the promoter for the
surfactant protein C is for the human surfactant protein C.
22. The method according to claim 9, wherein the expression vector
used in step a) in addition contains DNA of the SV40 virus.
23. The method according to claim 14, further comprising
cultivation of the removed cell tissue.
24. The utilization of a non-human mammal according to claim 1 for
the examination of the pathogenesis of lung tumors.
25. The utilization of a cell tissue according to claim 13 for the
examination of the pathogenesis of lung tumors.
26. A recombinant DNA expression vector containing a) the DNA
sequence of a constitutively active oncogenic mutant of the
kinase-domain of the c-Raf-1 gene or of a corresponding normal
allele or a derivative of the A, B or c-Raf-1 gene, b) a promoter
domain for the surfactant protein C, by means of which the
transcription of the DNA sequence defined in a) is controllable, c)
as an option the DNA sequence of the SV 40 virus
27. A recombinant DNA expression vector according to claim 26,
wherein the DNA sequence defined in a) is a sequence according to
FIG. 1 or a sequence .DELTA.Raf(26-302) derived therefrom, and/or
wherein the promoter domain defined in b) is a promoter domain for
the human surfactant protein c.
28. The utilization of a recombinant DNA expression vector
according to claim 26 for producing a transgenic mammal or a cell
tissue from such a mammal.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation under 35 U.S.C. .sctn.
120 of U.S. application Ser. No. 11/381,024 filed May 1, 2006,
which is a continuation under 35 U.S.C. .sctn. 120 of U.S.
application Ser. No. 10/371,138 filed Feb. 20, 2003, now abandoned,
which is a continuation under 35 U.S.C. .sctn. 120 of U.S.
application Ser. No. 09/555,279 filed Jul. 17, 2000, now U.S. Pat.
No. 6,566,581, which that claims the benefit of priority under 35
U.S.C. .sctn. 119 of 197 54 774.5 filed Nov. 28, 1997, the entire
contents of each is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a transgenic non-human mammal, to a
method for producing the latter, to the utilization thereof, to a
cell tissue therefrom, to a method for producing such cell tissue,
to the utilization thereof, to a recombinant DNA expression vector
and to the utilization of such vector. The term non-human mammal
refers to taxonomically higher units than animal species.
Transgenic animals are organisms carrying an additional gene not
originating from their species, that is a foreign gene in their
genome. For the purpose of the invention in particular, such
transgenic animals are meant that have the foreign gene also in the
germ cells, that is which hand on the foreign gene vertically, i.e.
from generation to generation. If a special transgenic animal has
been created, further corresponding transgenic animals may be
obtained by breeding. Transgenic animals are known in the art in
various embodiments, and various methods for producing transgenic
animals are also known. As an example only, reference is made to
document R. Jaenisch, Science, Vol. 240, 10, 1988, page 1468 ff.,
and the documents cited therein. The term cell tissue comprises
complete organs or parts of organs of an animal, however also
specific cell lines that can be isolated and cultivated therefrom,
i.e. increased in number.
[0003] A recombinant DNA expression vector is an instrument for
producing a transgenic animal carrying, among other features, the
foreign DNA to be integrated in the cells of the animal.
BACKGROUND OF THE INVENTION
[0004] The general technological background of the special
transgenic animal provided by the invention is the following.
Cancer, in particular lung cancer, is one of the most widespread
diseases of mankind, and has up to now therapy predictions offering
little success only. In the framework of the development of better
therapies for cancer diseases it is, among other conditions,
required and also legally laid down, for ethical reasons, to
perform pre-clinical examinations in animal models with possible
active substances obtained from basic research or by screening
tests. In the case of examinations of prospective active substances
for cancer therapies, it is therefore required to provide animals
or (animal) cell tissues having the respective cancer diseases to
be investigated, in order that the physiological effects, possibly
also side effects, of the active substances can be tested in a
qualitative and quantitative manner.
[0005] Cancer diseases are caused in many cases by the effects of
so-called oncoproteins. These are proteins that have different
structures compared to corresponding proteins in a healthy
organism. These oncoproteins are capable, through a not yet fully
understood processes, to transform normal cells into uncontrollably
proliferating cells, i.e. cancer cells. The formation of
oncoproteins in an organism is in turn caused by so-called
oncogenes, i.e. genes coding for the oncoprotein. Oncogenes may be
introduced into a cell by viruses, may however also be formed by
way of mutation of (certain) "healthy" genes, the proto-oncogenes.
Such mutations can for instance take place by translocation
(displacement) of a gene responsible for the production of a
protein within the genome, by point mutations (replacement of a
base and/or individual bases in the DNA of a gene responsible for
the production of a protein by a different base, with the
consequence of the formation of a protein of modified amino acid
sequence, the oncogene), by deletion (removal of one or more bases)
or also by mutations in the region of a so-called promoter
applicable for the respective gene. As a promoter is designated a
DNA region of a gene by means of which the transcription (of the
DNA code into a corresponding RNA) and thus finally also the
expression (formation) of the protein correlated with the gene can
be controlled. In a natural manner, a specific promoter is usually
assigned to each gene, this promoter being arranged ahead of the
latter in the genome. Ahead means that the promoter in the DNA
sequence has a certain distance to the starting point of a
transcription. For initiating a transcription, it is then also
required that so-called transcription factors (often specific for
the cell type) are taken up by the promoter.
[0006] In particular in connection with lung cancer, the so-called
Raf proto-oncogenes play a special role. These genes are highly
conservative with regard to evolution, and code kinases specific
for serine/threonine of the cytoplasm playing in turn a role in the
mitogenic signal transduction. Known in the art are for instance
the genes A, B and c-Raf-1. For a survey, reference is made to
documents U. R. Rapp et al., The Oncogene Handbook, Elsevier
Science Publishers, Netherlands, page 115-154, 1988, and U. R.
Rapp, Oncogene, 6, 495, 1991. To the family of the Raf genes
belongs, among others, the c-Raf-1 gene expressing the c-Raf-1
kinase ubiquitarily in an organism. The c-Raf-1 gene comprises
three conserved regions, i.e. these regions are in accordance with
corresponding regions of other Raf genes of the family. The region
CR1 is a regulatory domain around a cys finger consensus sequence,
the region CR2 is a region having a high content of serine or
threonine, and CR3 is the kinase domain. With regard to further
detailed information, reference is made to document U.S. Pat. No.
5,618,670. From this document are also known (partial) sequences of
the natural form of the CR3 region of the c-Raf-I gene of mice and
(partial) sequences of various point mutations thereof. From
document U.S. Pat. No. 5,156,841 are known plasmids and eucariotic
expression vectors containing A-Raf and v-Raf oncogenes, however in
different connections, namely the genic production of Raf
oncoproteins for immunological investigations.
SUMMARY OF THE INVENTION
[0007] The invention is based on the technical problem to provide
non-human mammals in sufficiently high numbers and with a pathology
being uniform and reproducible with regard to tumor formation, for
the purpose of pre-clinic examinations of prospective anti-cancer
substances or therapies.
[0008] For achieving this object, the invention teaches a
transgenic non-human mammal whose cells express a constitutively
active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or
a protein coded by a corresponding normal allele or a derivative of
the A, B or c-Raf-1 gene.
[0009] The term constitutively active means, in the context of the
invention, that the protein per se is always active, i.e. the
physiological effect of the protein is always obtained even without
the condition of further reaction cascades in a cell or an
organism. In contrast thereto, the activation of the not
constitutively active Raf-1 protein requires for instance the
bonding of the Raf protein with the Raf-1 protein. The term
constitutively active therefore refers, for the purpose of the
invention, only to the protein or the corresponding gene code and
not to the gene itself or the gene activation. The reference to the
Raf-1 gene means Raf-1 genes or variants thereof existing in any
organism, at least however such Raf-1 genes existing in mammals. By
the fact that the mammal is a transgenic animal having the
mentioned features, identical animals, with regard to the pathology
of the tumors induced by the expression of the mutant, can be
obtained in any number by way of the natural reproduction from a
transgenic base animal. Thereby pre-clinic examinations of active
substances or therapies can be performed with the required
reproducibility and the required statistical significance, and that
also with defined control groups.
[0010] For performing pre-clinic examinations of active substances
and therapies against lung cancer it is recommended that the
expression of the protein coded by the constitutively active
oncogenic mutant of the kinase-domain of the c-Raf-1 gene or by a
corresponding allele or a derivative of the A, B or c-Raf-1 gene
takes place in lung cells, since then the animals develop
reproducible lung tumors.
[0011] In structural regard, a mammal as described above is
characterized by that it contains foreign DNA with a constitutively
active oncogenic mutant of the kinase-domain of the c-Raf-1 gene or
with a corresponding normal allele or a derivative of the A, B or
c-Raf-1 gene. Alleles or derivatives are variants in a DNA sequence
virtually not affecting the basic function of the respective
gene.
[0012] Advantageously, the foreign DNA in addition contains a
promoter for the surfactant protein C, preferably for the human
surfactant protein C, and this promoter is arranged in the foreign
DNA with the proviso that by the promoter the transcription of the
mutant of the kinase-domain of the c-Raf-1 gene or of a
corresponding normal allele or a derivative of the A, B or c-Raf-1
gene is controlled in other words, the promoter being in a natural
manner arranged ahead of the gene coding the surfactant protein C,
is arranged instead, according to the invention, at a suitable
position of the mutant or of the gene. The precise arrangement of
promoter and mutant or gene with regard to each other is usual
knowledge of the man skilled in the art. If the exact positioning
of the special promoter ahead of the gene utilized according to the
invention cannot be derived from basic considerations, simple tests
with different variants of positioning can however be performed, in
order to determine a suitable position: The number of variants in
question, under consideration of the general technical knowledge,
is however very limited. The surfactant protein C plays a role for
the surfactant factor reducing the alveolar surface tension between
the lung epithelium and air and thus preventing that the alveoli
will collapse during breathing-out and that the epithelia will
stick together. By application of the promoter for the surfactant
protein C it is achieved that only the transcription factors
specifically or with increased frequency occurring in the lung and
inter-reacting with this promoter can so to speak switch on the
mutant, with the result that lung tumors will be formed at high
selectivity and reproducibility.
[0013] In a preferred further embodiment of the invention, the
foreign DNA in addition contains DNA of the SV40 virus. SV means
Simian Virus. This comprises the polyadenylated sequence and
intron/exon regions of the SV40 virus being known in the art (see
description of the following FIG. 1). The integration of this SV40
DNA causes an increase of the translation efficiency of the
polyadenylated mRNA. In detail it is preferred that the mammal
comes from the group of rodents.
[0014] A transgenic non-human mammal as described above is
obtainable by the following steps: a) integration of the cDNA
sequence of a constitutively active oncogenic mutant of the
kinase-domain of the c-Raf-1 gene or of a corresponding normal
allele or a derivative of the A, B or cRaf-1 gene in an expression
vector, b) insertion of the transgenic vector obtained in step a),
preferably after linearization, in pronuclei of fertilized oocytes
from a non-human mammal, c) implantation of the oocytes obtained in
step b) in brood animals of the same species as the donor species
of the oocytes and delivery of descendant animals from the oocytes,
d) genotypization and selection of the descendant animals obtained
in step c) with the proviso that cells of the selected mammals
express a constitutively active oncogenic mutant of the
kinase-domain of the c-Raf-1 gene or a protein coded by a
corresponding normal allele or a derivative of the A, B or c-Raf-1
gene. Genotypization can be obtained by means of methods well known
to the man skilled in the art, for instance by tail biopsy by means
of PCR (polymerase chain reaction, a method for the in-vitro
amplification of a defined DNA fragment) and Southern Blot (a
method for the analysis of DNA fragments in DNA preparations), with
those mammals being selected whose cells can be proven by the
examinations of the genotypization to contain the foreign DNA
according to the invention. Advantageously, the expression vector
used is step a) contains a promoter for the surfactant protein C,
preferably a promoter for the human surfactant protein C. This
promoter is arranged in the foreign DNA with the proviso that by
the promoter the transcription of the mutant of the kinase-domain
of the Raf-1 gene or of a corresponding normal allele or derivative
of the A, R or c-Raf-1 gene is controlled. In detail, the foreign
DNA may comprise for instance either the healthy (FIG. 1) or a
constitutively active oncogenic mutant of the Raf-1 gene with a
sequence according to one of FIG. 1, however with deletion as
.DELTA.Raf(26-302). In place of a deletion, point mutations of the
sequence shown in FIG. 1 can also be used. Such point mutations can
for instance specifically be caused by administration of
1-ethyl-1-nitrosourea (ENU) to animals having the healthy sequence,
thereby such mutants being accessible in a simple manner
[0015] The invention also relates to a method for producing a
transgenic non-human mammal as described above according to patent
claims 10 and 11. Non-human mammals according to the invention are
used for pre-clinic examinations of the effectiveness of substances
directed against lung carcinomas and/or therapeutical methods
directed against lung carcinomas, in particular for the pre-clinic
examination of the effectiveness of substances inhibiting
Raf-kinase. Such substances completely inhibit or reduce the
activity of Raf-kinases, thereby possibly a means for the
deactivation particularly of Raf-oncoproteins and therefore for the
proliferation inhibition of tumor cells being found. Another
advantageous utilization of a non-human, mammal according to the
invention is the investigation of the pathogenesis of lung tumors,
thereby a better understanding of the disease per se being
possible.
[0016] The invention however also relates to cell tissues, in
particular lung tissues, from a transgenic non-human mammal
according to one of claims 1 to 9, which cell tissue has a higher
probability of tumor formation, preferably of lung tumors, to a
method for the production thereof according to claims 16 and 17 and
to the utilization thereof according to claims 18 to 20. With
regard to the cell tissue or also specific cell lines comprised
therein, isolated and possibly cultivated therefrom, all general
explanations given above apply in corresponding manner.
[0017] Finally the invention also comprises a recombinant DNA
expression vector containing A) the DNA sequence of a
constitutively active oncogenic mutant of the kinase-domain of the
c-Raf-1 gene or of a corresponding normal allele or a derivative of
the A, B or c-Raf-1 gene, B) a promoter domain for the surfactant
protein C, by means of which the transcription of the DNA sequence
defined in A) is controllable, C) as an option the DNA sequence of
the SV40 virus. By means of such a vector the transgenic mammals
according to the invention and possibly also the cell tissues
therefrom can be produced. As an example, the DNA sequence defined
in A) comprises a sequence according to FIG. 4 or to the end of the
specification, respectively, or a sequence .DELTA.Raf(26-302)
derived therefrom, and/or the promoter domain defined in B) is a
promoter domain for the human surfactant protein C. The reproduced
sequence is that of human-c-Raf-1. Instead, also the c-Raf-1
sequences of the mouse, wild type or mutated, according to document
25 U.S. Pat. No. 5,618,670 can be used. Other sequences, even from
other organisms, are also possible, as far as they are basically a
Raf-1 sequence.
[0018] Subject matter of the invention is further a screening
method with utilization of the transgenic non-human mammals
according to the invention or of cell tissues therefrom, a group of
prospective active substances against cancer, in particular lung
cancer, being administered to the animals, and an evaluation of the
effects of each individual prospective active substance with regard
to proliferation inhibition, oncoprotein inhibition or the like is
performed. The invention further comprises active substances that
can be detected by such a screening method as being sufficiently
effective.
[0019] Plasmids with transgenic vectors according to the invention
(active oncogenic mutant of the kinase-domain of the human c-Raf-1
gene, i.e--plasmid SPC-.DELTA.Raf(26-302) and a transgenic vector
with normal human c-Raf-1 gene, i.e. plasmid SPC-Raf-1) were
registered at DSMZ--Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig. The
registration number for the plasmid SPC-.DELTA.Raf(26-302) is
11849. Thus the invention also relates to transgenic vectors as
registered, as well as to transgenic animals or cells or cell
tissues to be produced therefrom, and to the utilizations described
above of such animals or cells or cell tissues.
[0020] The various features of novelty which characterize the
invention are pointed out with particularity in the claims annexed
to and forming a part of this disclosure. For a better
understanding of the invention, its operating advantages and
specific objects attained by its uses, reference is made to the
accompanying drawings and descriptive matter in which preferred
embodiments of the invention are illustrated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] In the following, the invention will be described by figures
and embodiments representing examples only. There are:
[0022] FIG. 1 is a schematic representation of the structure of
vectors suitable for the invention,
[0023] FIG. 2 is a Southern Blot of selected transgenic animals
according to the invention,
[0024] FIGS. 3a-3b are Western or Northern Blots, respectively, of
obtained animal lines according to the invention, and
[0025] FIG. 4 is the DNA sequence (linear) of the kinase-domain of
the human-c-Raf-1 gene.
EXAMPLES [HUMAN Raf]
[0026] The general procedure during the production of transgenic
mice according to the invention was the following. The cDNA
sequences of the transforming c-Raf subdomain (Raf BxB) were cloned
into a lung-specific expression vector containing the promoter
region of the human surfactant-associated protein C(SPC). After
restriction digestion and linearization of these transgenic vectors
schematically shown in FIG. 2, the respective foreign DNA was
inserted into the pronuclei of fertilized oocytes, then implanted
into brood mice. The descendants of such mice were genotyped by
tail end biopsy by means of PCR and Southern Blot. By the thus
identified founder animals, mouse lines were established, in whose
lungs the expression of the transgene was detected in Western or
Northern Blot or by means of RT-PCR.
[0027] FIG. 1 shows lung-directed expression vectors for the
protein kinase Raf-1. The transgenic vectors include the 3.7 kb
(kilobases) wide region of the human SPC promoter, the 3.0 kb
c-Raf-1 or 1.4 kb human .DELTA.Raf(26-302) cDNA 10 fragments and a
0.4 kb fragment of viral DNA including the polyadenylation sequence
and intron/exon regions of the SV40 virus, thereby the translation
efficiency of the polyadenylated mRNA being increased. Further are
drawn the intersections of the used (also in the following
examples) restriction endonucleases.
[0028] The individual steps of the exemplary general procedure
described above are explained with regard to the experimental
measures in detail in the following examples.
Example 1
[0029] Cloning of the cDNA sequences of the transforming c-Raf
subdomain into a lung-specific expression vector was performed as
follows. The vector SPC-Raf-1 for generating the wild type-Raf-1
transgenic mice was produced by that a 3.0 kb fragment of the human
Raf-1 cDNA (Bonner, T. I.; Oppermann, R.; Seebrug, P.; Kerby, S.
B.; Gunnell, M. A.; Young, A. C.; and Rapp, U. R.; 1986; "The
complete coding sequence of the human raf oncogene and the
corresponding structure of the c-raf-1 gen."; Nucleic Acids Res.;
14, 109) was cloned in the EcoRI interface of the plasrid
SPC3.7/5V40 including the 3.7 kb promoter region of the human
surfactant-associated protein C(SPC) (Korfhagen, T. R.; Glasser, S.
W., Wert, S. E. Bruno, M. D.; Daugherty, C. C.; McNeish, J. D.;
Stock, J. L.; Potter, S. S.; Whitsett, J. A.; 1990; "Cis-acting
sequences from a human surfactant protein gene confer
pulmonary-specific gene expression in transgenic mice."; Proc.
Natl. Acad. Sci.; 87, 6122). In analogous manner, the transgenic
vector SPC-.DELTA.Raf(26-302) was cloned by insertion of a 1.4 kb
fragment, of human Raf-1 cDNA that has been obtained by deletion of
the amino acids 26 to 302 of the regulatory domain (Bruder, J. T.;
Heidecker, G.; and Rapp, U. R.; 1992; "Serum-, TPA-, and Rasinduced
expression from Ap-1/Ets-driven promoters requires Raf-1 kinase.";
Genes and Dev.; 6, 545) and thus contains the kinase-domain of the
Raf-1 protein (activated oncogenic mutant of the Raf-1 kinase).
Example 2
[0030] The linearization and the pronucleus injection were
performed as follows. The transgenic vectors were cut with the
restriction endonucleases NotI and NdeI, cleaned with a preparative
agarose gel (Sambrooks et al., 1989, see below), and diluted to a
concentration of 1 ng/ml. 200 ng of the linearized DNA fragments
were injected into the male pronuclei of fertilized oocytes.
Transgenic founder mice were identified by analysis of the genomic
DNA isolated from tail ends by Southern Blot (see also example 3).
The founder mice were crossed with non-transgenic B6D2 mice, in
order to establish stable lines.
[0031] The used mice were C57BL/6DBA F2 mice (B6D2 mice), and were
obtained from Harlan Winkelmann (Borchen) and from Charles River
(Sulzfeld), and were held and bred on in the stable of the MSZ
(Institut fur Medizinische Strahlenkunde and Zellforschung,
Wurzburg University, 097078 wurzburg) under pathogen-free
conditions.
Example 3
[0032] PCR and Southern Blot for genotypization were performed by
the following operational instructions. For the detection of the
transgenic integration, 10 .mu.g genomic DNA from tail ends were
cut over night with 40 units BamHI, separated on a 0.7% agarose gel
by means of electrophoresis, and transferred by means of a
capillary blot to nitrocellulose (Sambrook, J.; Fritsch, E. F.;
Maniatis, T.; "Molecular Cloning: a laboratory manual)"; Cold
Spring Harbor Laboratory Press). After fixation of the DNA by u.v.
light followed the detection of the transgene by hybridization of
the membrane (Church, G. M. and Gilbert, W.; "Genomic sequencing";
Proc. Natl. Acad. Sci.; 81, 1991) with a Raf-1 probe (containing
the Raf-1/SV40 sequence) that was obtained by digestion of the
transgenic vector SPC-Raf-1 with BamHI. The bound probe
cross-hybridizing also with the mouse-Raf locus, was detected by
exposure of the membrane on film material, and marked a 3.4 kb Raf
1/SV40 fragment or a 1.8 kb .DELTA.Raf (26-302)/SV40 fragment,
respectively. The result of Southern Blots from selected animals
according to the invention is shown in FIG. 2. Therein the
positions of endogenic Raf-1 and of the transgenic
.DELTA.Raf(26-302) or Raf-1 fragments in transgenic (T) and
non-transgenic (NT) mice for two independent mouse lines
(.DELTA.Raf.sub.--11 and .DELTA.Raf.sub.--23 or Raf 74 and Raf 87,
respectively) can be seen. The positions of the respective
fragments are identified by arrows. It can easily be found that the
bands to be assigned to transgenic .DELTA.Raf(26-302) or Raf-1
fragments, respectively (lower arrows), are only registered for the
transgenic animals.
Example 4
[0033] In total 2 c-Raf-BxB (or .DELTA.Raf(26-302), respectively)
mouse lines were obtained. These mice according to the invention of
both lines developed at an age of 6 to 7 months massive lung
carcinomas, the cellular origin of which corresponded with regard
to distribution pattern and pheno type to the pneumocytes type II.
Investigations of the time dependence of the tumor development
showed that after 2 months already significant multi-centric tumor
formation occurred. In the lung tissue of both lines could also be
detected the expression of the transgene in Western Blot, as
visible in FIG. 3. Thus also the correlation between specific
misapplied Raf kinase activity and lung tumor induction is proven
in vivo.
[0034] The Western Plot of FIG. 3 was obtained as follows. The
protein expression of the transgene was detected by that lung
tissue of the mice was lysated in detergent containing buffer, and
approx. 100 .mu.g of the solubilized proteins were split with a 10%
SDS polyacrylamid gel. After transfer of the proteins to
nitrocellulose, the membrane was incubated over night with
milk-powder containing blocking solution and successively for 1
hour each with a polyclonal anti-Raf-I rabbit anti-serum and with
peroxidace-coupled anti-rabbit immunoglobulin. After each
incubation step, several times washing solution was employed. The
detection of the bound anti-bodies took place by the reaction of
the peroxidase with a chemiluminescence emitting substrate and
exposure on film material. In FIG. 3 is shown in detail an immuno
blot with solubilized proteins from lung tissue of the specified
mouse lines and from non-transgenic control mice (C) afterstaining
with the anti-Raf rabbit anti-serum. Specified are the positions of
the respective Raf proteins at approx. 74 kDa (Raf-1) and 42 kDa
(.DELTA.Raf(26-302)). In FIG. 3a is identified the endogenic Raf-1
of the mouse and the .DELTA.Raf(26-302) in the transgenic mouse
lines. In FIG. 3b can be seen, due to the identical molecular
weight of endogenic murine Raf-1 and transgenic human Raf-1, the
transgenic expression caused by the increase in intensity of the
Raf-1 band.
[0035] The sequence listing in FIG. 4 is repeated in the following.
Sequence CWU 1
1
1 1 2977 DNA Human exon (105)..(105) exon (337)..(337) exon
(451)..(451) exon (553)..(553) exon (712)..(712) exon (811)..(811)
exon (963)..(963) exon (994)..(994) exon (1120)..(1120) exon
(1237)..(1237) exon (1324)..(1324) exon (1501)..(1501) exon
(1549)..(1549) exon (1667)..(1667) exon (1798)..(1798) exon
(1933)..(1933) 1 ccgaatgtga ccgcctcccg ctccctcacc cgccgcgggg
aggaggagcg ggcgagaagc 60 tgccgccgaa cgacaggacg ttggggcggc
ctggctccct cagg t ttaagaattg 115 tttaagctgc atcaatggag cacatacagg
gagcttggaa gacgatcagc aatggttttg 175 gattcaaaga tgccgtgttt
gatggctcca gctgcatctc tcctacaata gttcagcagt 235 ttggctatca
gcgccgggca tcagatgatg gcaaactcac agatccttct aagacaagca 295
acactatccg tgttttcttg ccgaacaagc aaagaacagt g g tcaatgtgcg 347
aaatggaatg agcttgcatg actgccttat gaaagcactc aaggtgaggg gcctgcaacc
407 agagtgctgt gcagtgttca gacttctcca cgaacacaaa ggt a aaaaagcacg
461 cttagattgg aatactgatg ctgcgtcttt gattggagaa gaacttcaag
tagatttcct 521 ggatcatgtt cccctcacaa cacacaactt t g ctcggaagac
gttcctgaag 573 cttgccttct gtgacatctg tcagaaattc ctgctcaatg
gatttcgatg tcagacttgt 633 ggctacaaat ttcatgagca ctgtagcacc
aaagtaccta ctatgtgtgt ggactggagt 693 aacatcagac aactctta t
tgtttccaaa ttccactatt ggtgatagtg gagtcccagc 752 actaccttct
ttgactatgc gtcgtatgcg agagtctgtt tccaggatgc ctgttagt t 811
ctcagcacag atattctaca cctcacgcct tcacctttaa cacctccagt ccctcatctg
871 aaggttccct ctcccagagg cagaggtcga catccacacc taatgtccac
atggtcagca 931 ccacgctgcc tgtggacagc aggatgattg a g gatgcaattc
gaagtcacag 983 cgaatcagcc t caccttcagc cctgtccagt agccccaaca
atctgagccc 1034 aacaggctgg tcacagccga aaacccccgt gccagcacaa
agagagcggg caccagtatc 1094 tgggacccag gagaaaaaca aaatt a ggcctcgtgg
acagagagat tcaagctatt 1150 attgggaaat agaagccagt gaagtgatgc
tgtccactcg gattgggtca ggctcttttg 1210 gaactgttta taagggtaaa tggcac
g gagatgttgc agtaaagatc ctaaaggttg 1267 tcgacccaac cccagagcaa
ttccaggcct tcaggaatga ggtggctgtt ctgcgc a 1324 aaacacggca
tgtgaacatt ctgcttttca tggggtacat gacaaaggac aacctggcaa 1384
ttgtgaccca gtggtgcgag ggcagcagcc tctacaaaca cctgcatgtc caggagacca
1444 agtttcagat gttccagcta attgacattg cccggcagac ggctcaggga atggac
t 1501 atttgcatgc aaagaacatc atccatagag acatgaaatc caacaat a
tatttctcca 1559 tgaaggctta acagtgaaaa ttggagattt tggtttggca
acagtaaagt cacgctggag 1619 tggttctcag caggttgaac aacctactgg
ctctgtcctc tggatgg c cccagaggtg 1677 atccgaatgc aggataacaa
cccattcagt ttccagtcgg atgtctactc ctatggcatc 1737 gtattgtatg
aactgatgac gggggagctt ccttattctc acatcaacaa ccgagatcag 1797 a
tcatcttcat ggtgggccga ggatatgcct ccccagatct tagtaagcta 1848
tataagaact gccccaaagc aatgaagagg ctggtagctg actgtgtgaa gaaagtaaag
1908 gaagagaggc ctctttttcc ccag a tcctgtcttc cattgagctg ctccaacact
1963 ctctaccgaa gatcaaccgg agcgcttccg agccatcctt gcatcgggca
gcccacactg 2023 aggatatcaa tgcttgcacg ctgaccacgt ccccgaggct
gcctgtcttc tagttgactt 2083 tgcacctgtc ttcaggctgc caggggagga
ggagaagcca gcaggcacca cttttctgct 2143 ccctttctcc agaggcagaa
cacatgtttt cagagaagct ctgctaagga ccttctagac 2203 tgctcacagg
gccttaactt catgttgcct tcttttctat ccctttgggc cctgggagaa 2263
ggaagccatt tgcagtgctg gtgtgtcctg ctccctcccc acattcccca tgctcaaggc
2323 ccagccttct gtagatgcgc aagtggatgt tgatggtagt acaaaaagca
ggggcccagc 2383 cccagctgtt ggctacatga gtatttagag gaagtaaggt
agcaggcagt ccagccctga 2443 tgtggagaca catgggattt tggaaatcag
cttctggagg aatgcatgtc acaggcggga 2503 ctttcttcag agagtggtgc
agcgccagac attttgcaca taaggcacca aacagcccag 2563 gactgccgag
actctggccg cccgaaggag cctgctttgg tactatggaa cttttcttag 2623
gggacacgtc ctcctttcac agcttctaag gtgtccagtg cattgggatg gttttccagg
2683 caaggcactc ggccaatccg catctcagcc ctctcaggag cagtcttcca
tcatgctgaa 2743 ttttgtcttc caggagctgc ccctatgggg cgggccgcag
ggccagcctg tttctctaac 2803 aaacaaacaa acaaacagcc ttgtttctct
agtcacatca tgtgtataca aggaagccag 2863 gaatacaggt tttcttgatg
atttgggttt taattttgtt tttattgcac ctgacaaaat 2923 acagttatct
gatggtccct caattatgtt attttaataa aataaattaa attt 2977
* * * * *