Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via retrovirus
Reexamination Certificate
1999-05-13
2004-05-11
Reynolds, Deborah J. (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via retrovirus
C800S021000, C800S004000
Reexamination Certificate
active
06734338
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts, particularly to the field of transgenics and gene therapy The invention is particularly directed to in vitro and in vivo methods for transfecting male germ cells and support cells (i.e., Leydig and Sertoli cells), which methods incorporate a method of depopulating a vertebrate testis of male germ cells.
2. Discussion of the Related Art
The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and phenomena of gene activation, expression, and interaction. This technology has been used to produce models for various diseases in humans and other animals. Transgenic technology is amongst the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function It is also used to study the relationship between genes and diseases About 5,000 diseases are caused by a single genetic defect. More commonly, other diseases are the result of complex interactions between one or more genes and environmental agents, such as viruses or carcinogens. The understanding of such interactions is of prime importance for the development of therapies, such as gene therapy and drug therapies, and also treatments such as organ transplantation Such treatments compensate for functional deficiencies and/or may eliminate undesirable functions expressed in an organism. Transgenesis has also been used for the improvement of livestock, and for the large scale production of biologically active pharmaceuticals.
Historically, transgenic animals have been produced almost exclusively by micro injection of the fertilized egg. The pronuclei of fertilized eggs are micro injected in vitro with foreign, i.e. xenogeneic or allogeneic DNA or hybrid DNA molecules. The micro injected fertilized eggs are then transferred to the genital tract of a pseudopregnant female. (E.g., P. J. A. Krimpenfort et al., Transgenic mice depleted in mature T-cells and methods for making transgenic mice, U.S. Pat. Nos. 5,175,384 and 5,434,340; P. J. A. Krimpenfort et al., Transgenic mice depleted in mature lymphocytic cell-type, U.S. Pat. No. 5,591,669).
The generation of transgenic animals by this technique is generally reproducible, and for this reason little has been done to improve on it. This technique, however, requires large numbers of fertilized eggs. This is partly because there is a high rate of egg loss due to lysis during micro injection. Moreover manipulated embryos are less likely to implant and survive in utero. These factors contribute to the technique's extremely low efficiency. For example, 300-500 fertilized eggs may need to be micro injected to produce perhaps three transgenic animals. Partly because of the need to micro inject large numbers of embryos, transgenic technology has largely been exploited in mice because of their high fecundity. Whilst small animals such as mice have proved to be suitable models for certain diseases, their value in this respect is limited. Larger animals would be much more suitable to study the effects and treatment of most human diseases because of their greater similarity to humans in many aspects, and also the size of their organs. Now that transgenic animals with the potential for human xenotransplantation are being developed, larger animals, of a size comparable to man will be required. Transgenic technology will allow that such donor animals will be immunocompatible with the human recipient. Historical transgenic techniques, however, require that there be an ample supply of fertilized female germ cells or eggs. Most large mammals, such as primates, cows, horses and pigs produce only 10-20 or less eggs per animal per cycle even after hormonal stimulation. Consequently, generating large animals with these techniques is prohibitively expensive.
This invention relies on the fact that vast numbers of male germ cells are more readily available. Most male mammals generally produce at least 10
8
spermatozoa (male germ cells) in each ejaculate. This is in contrast to only 10-20 eggs in a mouse even after treatment with superovulatory drugs. A similar situation is true for ovulation in nearly all larger animals. For this reason alone, male germ cells will be a better target for introducing foreign DNA into the germ line, leading to the generation of transgenic animals with increased efficiency and after simple, natural mating.
Spermatogenesis is the process by which a diploid spermatogonial stem cell provides daughter cells which undergo dramatic and distinct morphological changes to become self-propelling haploid cells (male gametes) capable, when fully mature, of fertilizing an ovum.
Primordial germ cells are first seen in the endodermal yolk sac epithelium at E8 and are thought to arise from the embryonic ectoderm (A. McLaren and Buehr, Cell Diff. Dev. 31:185 [1992]; Y. Matsui el al., Nature 353:750 [1991]). They migrate from the yolk sac epithelium through the hindgut endoderm to the genital ridges and proliferate through mitotic division to populate the testis.
At sexual maturity the spermatogonium goes through 5 or 6 mitotic divisions before it enters meiosis. The primitive spermatogonial stem cells (Ao/As) proliferate and form a population of intermediate spermatogonia types Apr, Aal, A1-4 after which they differentiate into type B spermatogonia. The type B spermatogonia differentiate to form primary spermatocytes which enter a prolonged meiotic prophase during which homologous chromosomes pair and recombine. The states of meiosis that are morphologically distinguishable are; preleptotene, leptotene, zygotene, pachytene, secondary spermatocytes and the haploid spermatids. Spermatids undergo great morphological changes during spermatogenesis, such as reshaping the nucleus, formation of the acrosome and assembly of the tail (A. R. Bellve et al,
Recovery, capacitation, acrosome reaction, and fractionation of sperm
, Methods Enzymol. 225:113-36 [1993]). The spermatocytes and spermatids establish vital contacts with the Sertoli cells through unique hemi-junctional attachments with the Sertoli cell membrane. The final changes in the maturing spermatozoan take place in the genital tract of the female prior to fertilization.
Initially, attempts were made to produce transgenic animals by adding DNA to spermatozoa which were then used to fertilize mouse eggs in vitro. The fertilized eggs were then transferred to pseudopregnant foster females, and of the pups born, 30% were reported to be transgenic and express the transgene. Despite repeated efforts by others, however, this experiment could not be reproduced and no transgenic pups were obtained. Indeed, there remains little doubt that the transgenic animals reputed to have been obtained by this method were not transgenic at all and the DNA incorporation reported was mere experimental artifact. Data collected from laboratories around the world engaged in testing this method showed that no transgenics were obtained from a total of 890 pups generated.
In summary, it is currently possible to produce live transgenic progeny but the available methods are costly and extremely inefficient. Spermatogenic transfection in accordance with this invention, either in vitro or in vivo, provides a simple, less costly and less invasive method of producing transgenic animals and one that is potentially highly effective in transferring allogeneic as well as xenogeneic genes into the animal's germ cells.
To facilitate in vitro transfection of male germ cells and implantation into a testis of a recipient male vertebrate it is advantageous first to depopulate the testis of the recipient vertebrate of untransfected male germ cells before trans
Readhead Carol W.
Winston Robert
Cedars-Sinai Medical Center
Reynolds Deborah J.
Sidley Austin Brown & Wood LLP
Woitach Joseph
LandOfFree
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