Transfection, storage and transfer of male germ cells for generation of transgenic species & genetic therapies

Abstract

Disclosed is an in vivo method of incorporating exogenous genetic material into the genome of a vertebrate, which involves administering to a male vertebrate's testis a gene delivery mixture comprising a viral vector, such as a retroviral vector, to deliver a polynucleotide encoding a desired trait or product. Also disclosed is an in vitro method of incorporating exogenous genetic material into the genome of a vertebrate, in which germ cells are obtained from a donor male vertebrate and are genetically modified in vitro, before being transferred to a recipient male vertebrate. After the transfer, the male vertebrate bearing the genetically modified germ cells is bred with a female vertebrate such that a transgenic progeny is produced that carries the polynucleotide in its genome. Also disclosed are non-human transgenic vertebrates produced in accordance with the method, including transgenic progeny. A transgenic cell derived from the transgenic vertebrate is also disclosed, being a germ cell, such as a spermatozoan or ovum, a precursor cell of either of these, or a somatic cell. A method of producing a non-human transgenic vertebrate animal line comprising native germ cells carrying in their genome at least one xenogeneic polynucleotide is disclosed, as is vertebrate semen containing the transgenic male germ cells useful in practicing the method.

Description

[0001]This application is a continuation-in-part of U.S. Ser. No. 09 / 311,599, filed May 13, 1999, which is a continuation-in-part of U.S. Ser. No. 09 / 191,920, filed Nov. 13, 1998. This application claims the benefit of U.S. Provisional Application No. 60 / 065,825 which was filed on Nov. 14, 1997.BACKGROUND OF THE INVENTION[0002]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.[0003]1.The Field of the Invention[0004]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 genetically modifying male germ cells and support cells (i.e., Leydig and Sertoli cells), which methods incorporate a method depopulating a vertebrate testis of male...

AI Technical Summary

Benefits of technology

[0028]The present invention relates to the in vivo and in vitro (ex vivo) genetic modification, for example, by transfection or transduction, of vertebrate animal germ cells with a desired genetic material. Briefly, the in vivo method involves injection of genetic material together with a suitable vector directly into the testicle of the animal. In this method, all or some of the male germ cells within the-testicle are genetically Emodified in situ, under effective conditions. The in vitro method involves obtaining germ cells from the gonad (i.e., testis) of a suitable donor or from the animal's own testis, using a novel isolation or selection method, transfecting or otherwise genetically altering them in vitro, and then returning them to the substantially depopulated testis of the donor or of a different recipient male vertebrate under suitable conditions where they will spontaneously repopulate the depopulated testis. The in vitro method has the advantage that the transfected germ cells can be screened by various means before being returned to the testis of the same or a different suitable recipient male to ensure that the transgene is incorporated into the genome in a stable state. Moreover, after screening and cell sorting only enriched populations of germ cells can be returned. This approach provides a greater chance of transgenic progeny after mating.

Problems solved by technology

These prior methods are extremely slow and costly, rely on several invasive steps, and only produce transgenic progeny sporadically and unpredictably.

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