Transgenic production from in vivo-derived embryos: effect on calf birth weight and sex ratio.
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.
Behboodi
E
E
Genzyme Transgenics Corporation, Framingham, Massachusetts 01701-9322, USA.
Groen
W
W
Destrempes
M M
MM
Williams
J L
JL
Ohlrichs
C
C
Gavin
W G
WG
Broek
D M
DM
Ziomek
C A
CA
Faber
D C
DC
Meade
H M
HM
Echelard
Y
Y
D000818:Animals; D030801:Animals, Genetically Modified; D001724:Birth Weight / Q000235:genetics*; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D004624:Embryo Transfer / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000196:embryology* / Q000378:metabolism*; D005260:Female; D005307:Fertilization in Vitro; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008845:Microinjections; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics; D012661:Semen / Q000378:metabolism; D019849:Sex Determination Processes; D012744:Sex Ratio; D019076:Transgenes / Q000235:genetics
D004247:DNA