EXPRESSION OF 2 TRANSGENES IN IN-VITRO MATURED AND FERTILIZED BOVINE ZYGOTES AFTER DNA MICROINJECTION

Bovine zygotes produced by in vitro matrration-in vitro fertilization (IVM-IVF) were examined for their potential to serve as recipients of transgenes. Pronuclei, which were maximally visible at about 22 h afte r IVF, were injected with a SV40-LacZ construct (pSVON). Injected zygo tes had lower cleavage rates (49.1%, n = 1162, P < 0.01) than did eith er noninjected controls (87.4%, n = 1420) or noninjected zygotes in wh ich pronuclei were not visible (67.6%, n = 803). Zygotes that were inj ected into their pronuclei cleaved as well as zygotes injected cytopla smically. At 48 h after injection, when most embryos had reached the f our- and eight-cell stages, more zygotes in the pronuclear group (22.7 %, n = 125) stained positively for LacZ than did zygotes in the cytopl asmic group (8.0%, n = 125). A group of zygotes injected into the pron ucleus with pSVON was cultured for 9 days. More morulae (10.8%, n = 13 4) than blastocysts (3.2%, n = 31) expressed the LacZ gene, indicating that silencing of expression occurred as development progressed. Anot her group of zygotes was injected with a beta-actin-LacZ gene construc t (pbActinLacZ) and, of the embryos assayed at 48 h 10.6% (n = 255) st ained positively. At 9 days, 36.3% of morulae (n = 91) and 21% of blas tocysts (n = 33) expressed the transgene. Almost all putative transgen ic embryos injected with either construct showed a mosaic pattern of L acZ expression, with an average of only 2-3 cells staining at the eigh t-cell stage and the majority of cells in positive blastocysts showing no evidence of expression.