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Transfection, storage and transfer of male germ cells for generation of trangenic species

a technology of trangenic species and germ cells, applied in the field of transgenics and gene therapy, can solve the problems of germ cells being genetically modified or no

Inactive Publication Date: 2002-09-12
WINSTON ROBERT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] The present invention relates to the in vivo and ex vivo (in vitro) transfection of eukaryotic 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 transfected in situ, under effective conditions. The ex vivo method involves extracting germ cells from the gonad of a suitable donor or from the animal's own gonad, using a novel isolation method, transfecting them in vitro, and then returning them to the testis under suitable conditions where they will spontaneously repopulate it. The ex vivo method has the advantage that the transfected germ cells may be screened by various means before being returned to the testis 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 may be returned. This approach provides a greater chance of transgenic progeny after mating.

Problems solved by technology

These germ cells may or may not be genetically manipulated prior to reimplantation.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0047] Preparation of Transferrin-Poly-L-Lysine Complexes

[0048] Human transferrin was conjugated to poly (L-lysine) using EDC (1-ethyl-3-(3-dimethyl aminopropyl carbodiimide hydrochloride) (Pierce), according to the method of Gabarek and Gergely (Gabarek & Gergely, Zero-length cross-linking procedure with the use of active esters, Analyt. Biochem 185 : 131 (1990)). In this reaction, EDC reacts with a carboxyl group of human transferrin to form an amine-reactive intermediate. The activated protein was allowed to react with the poly (L-lysine) moiety for 2 hrs at room temperature, and the reaction was quenched by adding hydroxylamine to a final concentration of 10 mM. The conjugate was purified by gel filtration, and stored at -20.degree. C.

example 2

[0049] Preparation of DNA for In Vivo Trasfection

[0050] The Green Lantern-1 vector (Life Technologies, Gibco BRL, Gaithersberg, Md.) is a reporter construct used for monitoring gene transfection in mammalian cells. It consists of the gene encoding the Green Fluorescent Protein (GFP) driven by the cytomegalovirus (CMV) immediate early promoter. Downstream of the gene is a SV40 polyadenylation signal. Cells transfected with Green Lantern-1 fluoresce with a bright green light when illuminated with blue light. The excitation peak is 490 nm.

example 3

[0051] Preparation of Adenoviral Particles

[0052] Adenovirus dI312, a replication-incompetent strain deleted in the Ela region, was propagated in the Ela trans-complementing cell line 293 as described by Jones and Shenk (Jones and Shenk, PNAS USA (1979) 79: 3665-3669). A large scale preparation of the virus was made using the method of Mittereder and Trapnell (Mittereder et al., "Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy", J. Urology, 70: 7498-7509 (1996)). The virion concentration was determined by UV spectroscopy, 1 absorbance unit being equivalent to 10 viral particles / ml. The purified virus was stored at -70.degree. C.

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PUM

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Abstract

A composition for in vivo transfection of vertebrate male germ cells comprises a nucleic acid or transgene, and a gene delivery system, and optionally a protective internalizing agent, such as an endosomal lytic agent, a virus or a viral component, which is internalized by cells along with the transgene and which enhances gene transfer through the cytoplasm to the nucleus of the male germ cell. A pharmaceutical preparation and a transfer kit utilize the composition. A method for introducing a polynucleotide into vertebrate male germ cells comprises the administration of the composition to a vertebrate. A method for isolating or selecting transfected cells utilizes a reporter gene, and a method for administering transfected male germ cells utilizes male germ cells which have been transfected in vitro.

Description

BACKGROUND OF THE INVENTION[0001] 1. Field of the Invention[0002] The present invention relates to the field of transgenics and gene therapy. More specifically, this invention relates to in vitro and in vivo methods for transfecting germ cells and, in some instances, incorporating a nucleic acid segment encoding a specific trait into the male germ cells of an animal. When the nucleic acid becomes incorporated into the germ cell genome, upon mating, or in vitro fertilization and the like, the trait may be transmitted to the progeny. The present technology is suitable for breeding progeny with or without a desired trait by modifying their genome. This technology is also suitable for use in introducing a therapeutic gene into the germ or support cells (e.g., Leydig and Sertoli cells) of the testis and is, therefore, suitable for use in gene therapy for males with fertility problems associated with genetic defects.[0003] 2. Description of the Background[0004] The field of transgenics wa...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A61K35/12A61K35/48A61K45/00A61K48/00A61P15/08A61P43/00C12N5/076C12N5/10C12N15/09C12N15/85C12Q1/68
CPCA01K67/027A01K67/0275A01K2217/05A01K2227/10A01K2227/105A01K2267/01A01K2267/02A01K2267/025A01K2267/03A61K35/12A61K48/00C12N5/061C12N15/8509C12N2510/00C12N2510/02C12N2799/021C12N2799/022A61P15/08A61P43/00
Inventor WINSTON, ROBERTREADHEAD, CAROL W.
Owner WINSTON ROBERT
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