Gene transfer to mammalian zygotes

Abstract

A total of 302 one-celled ova were surgically collected from the oviducts of 65 superovulated crossbred heifers 30-72 h post onset of estrus. Two hundred and sixty-two one-celled ova which were collected from 50 heifers 48 h post onset of estrus were treated with 4'-6'-diamidino-2-phenylindole and observed under ultraviolet light by fluorescence microscopy. Both male and female pronuclei were visualized in 131 zygotes. The male pronucleus of 85 zygotes was injected with 1-2 picolitres (300-500 copies) of plasmid pBR322 containing the Herpes Simplex Virus Thymidine Kinase gene. Forty-one gene injected zygotes were transferred to the oviducts of 11 synchronized recipients. Two recipients have been determined to be pregnant by palpation per rectum and ultrasonography. Twenty-three gene injected zygotes were transferred to the oviducts of three recipients which were used as in vivo incubators. A 39% recovery rate on reflushing of the reproductive tracts was obtained. Embryonic growth was observed in 7 of the 9 reflushed embryos varying from the two-celled to the eight-celled stage. Six of fifteen (40%) in vitro incubated gene injected zygotes developed further in culture.Intracytoplasmic injection of liposomes encapsulating HSV-TK gene into 60 murine zygotes resulted in liposome-pronuclear membrane apposition in seven zygotes and the development of one zygote to the two-celled stage in culture. These preliminary results suggest that liposome-mediated gene transfer may be developed as a new vector system for gene transfer to mammalian zygotes. Microinjection of genes into pronuclei of bovine zygotes and the subsequent development of injected zygotes both in vitro and in vivo in this study, renders support to the possibility that direct microinjection of genes into early embryos of farm animal species may be an appropriate approach to the production of transgenic livestock. Liposome encapsulated genes possess the potential for application as an alternate approach to pronuclear injection. This study presents data which strengthen the feasibility of transgenic livestock production.