Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

^*Endocrinology-Reproductive Physiology Program, University of Wisconsin, 1675 Observatory Drive, Madison, WI 53706; ^†Gala Design LLC, P.O. Box 520, Sauk City, WI 53583-0520; and ^§University of California-San Diego School of Medicine, Department of Pediatrics/0830, 9500 Gilman Drive, La Jolla, CA 92093-0830

Communicated by Neal L. First, University of Wisconsin, Madison, WI (received for review December 15, 1997)

Abstract

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

Footnotes

↵ ‡ Present address: Oregon Regional Primate Research Center, Beaverton, OR 97006.
↵ ¶ To whom reprint requests should be addressed. e-mail: Robert_Bremel{at}gala.com.
ABBREVIATIONS:

MII,

metaphase II;

VSV-G,

vesicular stomatitis virus envelope glycoprotein G;

RSV,

Rous sarcoma virus;

LTR,

long terminal repeat;

HbsAg,

hepatitis B surface antigen;

PSI,

perivitelline space injection;

X-gal,

5-bromo-4-chloro-3-indolyl β-d-galactoside