Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-03-22
2002-06-25
Wilson, Michael C. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S008000, C800S013000, C800S021000, C435S325000, C435S455000
Reexamination Certificate
active
06410825
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to transgenic non-human animals and transgenic non-human animal cells harboring a transgene containing a mutation in the A-myb gene and having a functionally disrupted A-myb gene locus. The invention further relates to transgenes and targeting constructs used to produce such transgenic animals and cells, methods of using such animals for modeling male infertility disorders, and methods for using such animals to produce transgenic nonhuman animals and cells including a further transgene.
BACKGROUND OF THE INVENTION
The myb gene family currently consists of three members, named A, B and c-myb. Of these, c-myb is the most extensively studied member. The B-myb and A-myb genes share extensive sequence homology with c-myb.
The myb oncogene was first identified as the transforming gene of Avian Myeloblastosis virus (AMV) which causes myeloblastic leukemia in chickens and transforms myelomonocytic cells in culture (Baluda et al.,
Virology
15: 185-199 (1964); C. Moscovici,
Immunol
. 71: 79-101 (1975)). The normal cellular counterpart of this oncogene, c-myb, is highly conserved and is present in all vertebrate and some invertebrate species examined (Franchini et al.,
Proc. Nat. Acad. Sci. USA
80: 7385-7389 (1983); Katzen et al., Cell 41: 449-456 (1985)). Proteins encoded by the viral as well as the cellular myb gene appear to be localized in the nucleus, and these proteins exhibit a sequence-specific DNA-binding activity (Klempnauer et al.,
Cell
37: 537-547 (1984); Boyle et al.,
Proc. Nat. Acad. Sci. USA
81: 42654269 (1984); Moelling et al.,
Cell
40: 983-990 (1985); Biedenkapp et al.,
Nature
335: 835-837 (1988)). Their sequence-specific DNA binding activity and ability to activate transcription of reporter genes linked to certain promoter/enhancer sequences suggest that they act as nuclear transcription factors (Sakura et al.,
Proc. Nat. Acad. Sci. USA
86: 5758-5762 (1989); Dudek et al.,
Proc. Nat. Acad. Sci. USA
89: 1291-1295 (1992)). A-myb in particular has been recognized as a potent transactivator of transcription (Golay et al.,
Oncogene
9: 2469-2479 (1994); Foos et al.,
Oncogene
9: 2481-2488 (1994)). Elimination of c-myb function in vivo, using gene-knock out techniques, has indicated that homozygous c-myb mutant mice fail to show effective fetal hepatic hematopoiesis resulting in the death of mice in utero confirming an essential role for c-myb in fetal hematopoiesis (Mucenski et al.,
Cell
65: 677-689 (1991)).
In contrast to c-myb, whose role in hematopoiesis is well established, little is known about the role of the A-myb gene in development. Human A-myb is expressed in a variety of lymphoid and solid tumors (Shen-Ong et al.,
Mol. Cell. Biol
. 6: 380-392 (1986)). Foos et al.,
Oncogene
9: 2481-2488 (1994) have reported ubiquitous expression of A-myb in chicken cell lines. On the other hand, Sleeman,
Oncogene
8: 1931-194 (1993) reported specific expression of Xenopus A-myb in testis, with very low levels of expression in ovarian tissue.
Murine spermatogenesis is divided into three distinct intervals which include: (1) stem cell proliferation and renewal; (2) meiosis and (3) germ cell differentiation (spermatogenesis). Spermatogenesis in mice occurs in the seminiferous tubule, a specialized epithelium in which spermatogonia are located in close proximity to the basement membrane. Cells at progressively later stages of meiosis and differentiation are situated closer to the tubular lumen. Spermatogenesis in the mouse occurs in twelve distinct histological stages. Each stage consists of a constant pattern of germ cell association. Stage VII of mouse spermatogenesis is a testosterone dependent stage and includes the following cell types: Type A spermatogonia (stem cells) along with preleptotene spermatocytes, usually situated closest to the basement membrane; pachytene spermatocytes (early meiotic cells) located at the intermediate position between basement membrane and the lumen; and step 7 spermatids and step 16 spermatozoa located closest to the lumen.
Recently, it has been demonstrated that A-myb is expressed at high levels in mouse testis where it is transcribed as multiple transcripts, some of which are differentially spliced to code for smaller proteins (Mettus et al., Oncogene 9: 3077-3086 (1994)). A high level of expression of A-myb was seen in mouse testis and very low levels of expression were detected in mouse spleen, ovary and brain. As differentiation proceeds and the primary spermatogonia mature into secondary spermatogonia, which in turn maturate into spermatocytes, a distinct downregulation of A-myb expression was seen in in Situ hybridization studies. A-myb was maximally expressed in type A spermatogonia which are located proximal to the basement membrane and preleptotene and pachytene spermatocytes located between the basement membrane and the lumen. Less intense hybridization was also seen with spermatids. Thus, A-myb expression was maximal in proliferating stem cells and early meiotic cells but reduced in spermatids and absent in spermatozoa undergoing terminal differentiation.
Despite these findings, the functional significance of A-myb remains to be established, particularly in spermatogenesis. More complete information concerning the function of A-myb requires studying the effect of the encoded protein, or the lack thereof, in vivo.
Various animals have been produced with germ line foreign DNA, or with altered levels of expression of certain genes. These animals typically have a foreign or mutated gene incorporated into their genome. In one such class of transgenic animal, the so-called homozygous null or “knockout” mutants, expression of an endogenous gene has been suppressed through genetic manipulation.
Transgenic animals generally harbor at least one copy of a transgene either homologously or nonhomologously integrated into an endogenous chromosomal location so as to encode a foreign or mutant protein. Such transgenic animals are usually produced by introducing the transgene or targeting construct into a fertilized egg, or into an embryonic stem (ES) cell which is then injected into an embryo. Introduction of the transgene into the fertilized egg or ES cell is typically performed by microinjection, retroviral infection, electroporation, lipofection, or biolistics. The fertilized egg or embryo is then transferred to an appropriate pseudopregnant female for the duration of gestation. Knockout mutants may be obtained according to this method where the non-native DNA which is introduced comprises a nucleic acid construct that will be used to suppress expression of a particular gene. Such knockout constructs are typically introduced into ES cells.
One problem in the production of transgenic animals is the relatively low rate of success in obtaining incorporation of the transgene into the germline of the host species. Moreover, while transgenes have been incorporated into fertilized eggs by microinjection, the smaller size of sperm cells makes incorporation of transgenes by injection difficult. What is needed is a method to increase the frequency of first generation transgenic offspring and to provide for the incorporation of transgenes into sperm.
Male infertility continues to be significant reproductive health problem. What is needed is a live animal model which may be used for the study of male infertility, and for screening and evaluation of potential therapeutic agents useful in the treatment of this disorder.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the invention to provide nonhuman animals in which expression of the A-myb gene has been suppressed.
It is an object of the invention to provide nonhuman cells and nonhuman animals containing a homozygous null mutation of the A-myb gene locus.
It is an object of the invention to provide constructs and vectors for producing such cells and animals containing an A-myb homozygous null mutation.
It is a further object of the invention to provide a method for obtaining incorporation of transgenes of interest into sperm cells, and
Hatton Kimi
Reddy E. Premkumar
Toscani Amelia
Toscani Antonio
Toscani Donato
Drinker Biddle & Reath LLP
Temple University-Of The Commonwealth System of Higher Education
Toscani Amelia
Toscani Donato
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