Behboodi E, Groen W, Destrempes M M, Williams J L, Ohlrichs C, Gavin W G, Broek D M, Ziomek C A, Faber D C, Meade H M, Echelard Y
Genzyme Transgenics Corporation, Framingham, Massachusetts 01701-9322, USA.
Mol Reprod Dev. 2001 Sep;60(1):27-37. doi: 10.1002/mrd.1058.
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.
我们通过将DNA显微注射到1细胞、2细胞和4细胞胚胎中来研究转基因牛的生产，分析其对犊牛大小和后续生存能力的影响。胚胎要么是在屠宰场通过冲洗经超数排卵和人工授精的母牛的输卵管收集（体内来源），要么是通过对从切除的卵巢中吸出的卵母细胞进行体外成熟和体外受精获得（体外来源）。将人血清白蛋白（hSA）乳汁表达DNA构建体显微注射到体外和体内来源的1细胞胚胎的一个可见原核中，或者注射到体内来源的2细胞和4细胞胚胎的两个卵裂球的细胞核中。所有显微注射胚胎的显微注射诱导死亡率（裂解和发育阻滞）相当（约40%）。胚胎在含有10%胎牛血清（FSC）的B-2培养基中与BRL细胞共培养。总体而言，体内来源的卵子发育到桑葚胚/囊胚的比例（15.5%）显著高于体外来源的卵母细胞（9.3%）。所有桑葚胚和囊胚在受精后第6 - 8天被移植到同期受体母畜体内。共分娩了189头犊牛。与体内来源的卵母细胞产生的犊牛相比，体外来源的卵母细胞产生的犊牛出生体重显著更高。通过对一个2细胞胚胎进行显微注射获得了一头转基因公牛犊。对淋巴细胞的荧光原位杂交（FISH）分析在所有细胞中检测到一个转基因整合位点。利用转基因犊牛的精液通过IVM/IVF/IVC获得的胚胎中hSA转基因的传递频率证实它不是嵌合体。
