Transgenic production from in vivo‐derived embryos: Effect on calf birth weight and sex ratio

Abstract

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.

The use of green fluorescent protein (GFP) to select bovine embryos

“…Corresponding results for our previous studies were 44 and 32.2% (Duszewska et al, 2000;Rosochacki et al, 2001). The greater negative effect on embryo development in the present study may be due to the increase in microinjected DNA concentration (3 ng in our previous study vs 5 ng in this one), an observation that was also shown by Behboodi et al (2001). It should be pointed out, that the development of compacted morulae and blastocysts from 48 h cleaved embryos was very similar, being 48% in both groups.…”

“…A higher percentage of morulae and blastocysts was obtained by Wang et al (2001) but they also obtained a higher percentage of viable zygotes after injection (83%). The yield of morulae/blastocysts resulting from 1-cell in vivo derived embryos that had survived microinjection could have been even 50% higher than from equivalent in vitro-derived embryos (Krimpenfort et al, 1991;Behboodi et al, 1993;Behboodi et al, 2001). In our work we used only in vitro derived oocytes.…”

“…In the work of Behboodi et al (2001) 2578 in vitro fertilized 1-cell embryos were microinjected but no transgenic calves were born. However, they did obtained one transgenic bull calf from the microinjection 8193 of two-cell in vivo fertilized embryos.…”

Obtaining calves after transfer of embryos microinjected with the human interferon alpha (IFNα) gene, pbLGIFN-GFPBsd

“…The results of the present study support this opinion in that, after microinjection, development to the blastocyst stage was only 10.4% compared with 39.9% for non-injected embryos (Table 1). This result is comparable with other studies in which the effi ciency of embryo development to the blastocyst stage ranged between 6 and 14% (Eyestone, 1999;Behboodi et al, 2001). In our experiment, the negative effect of transgene introduction occurred in the fi rst cell cycle because the microinjected embryos developed better (61.1%) from cleavage to the blastocyst stage than the control embryos (48.6%).…”

Production of Transgenic Bovine Cloned Embryos Using Piggybac Transposition

“…Furthermore, this technique has stimulated interest in developing transgenic cattle, resulting in production of several transgenic cloned cattle. Initially, transgenesis in cattle was carried out by microinjecting DNA into pronuclear stage oocytes [1], as in rabbits, sheep and pigs [10]. After examining somatic cell nuclear transfer (SCNT) as a better approach to achieve transgenesis in cattle [21], donor cells containing target genes were reprogrammed in enucleated oocytes and produced transgenic calves [3,17,23].…”