注入嵌合法制作黄牛和水牛种间嵌合胚的初步研究

Reconstruction of interspecific chimeric embryos between cattle and buffalo by ICM injection

采用注入嵌合法初步建立了一套黄牛和水牛种间嵌合的程序与方法。采用机械剥离法或免疫外科法分离胚胎内细胞团(ICM),然后注入到已去除ICM的受体囊胚中构建形成水牛和黄牛的嵌合胚。结果发现,在用免疫外科法分离ICM时,抗血清的灭活温度从57℃升至63.5℃,ICM的获得数显著升高(0%vs100%,P<0.01),如若在分离培养液中添加6%的胎牛血清(FCS),ICM的获得数大大降低(97.6%vs0%,P<0.01)。采用免疫外科法分离得到的黄牛ICM进行水牛囊胚的ICM置换重组,重组胚的存活率与机械剥离法得到的ICM无显著差异(91.4%vs87.5%,P>0.05);但囊胚孵化率则显著提高(80%vs43.8%;P<0.05)。以上结果表明,⑴水牛和黄牛胚胎通过ICM置换获得的种间嵌合胚胎能继续发育;⑵用于黄牛ICM分离的兔抗牛抗血清需在63.5℃灭活30min,且分离需在无血清的培养液中进行;⑶通过分离ICM置换进行胚胎嵌合时,免疫外科法优于显微手术法。

In order to establish a preliminary procedure for reconstructing interspecific chimeras between cattle and buffalo by inner cell mass （ICM） injection, bovine ICM was isolated from blastocysts by either mechanical or immunosurgical method, and injected into buffalo blastocysts that ICM was removed to reconstruct a chimeric embryo. The results showed that more ICM were isolated from bovine blastocysts by immunosurgery when the anti-cattle rabbit serum was inactivated at 63.5 ℃ than that at 56℃（ 100% vs 0%, P 〈0. 01 ）. Supplement of 6% FCS in isolated medium resulted in a significant decrease in ICM isolating efficiency （0% vs 97. 6%, P 〈0.01 ）. More chimeric buffalo blastocysts hatched when their ICM were replaced with bovine ICM isolated by immunosurgery （80%） compared with the ICM isolated by microsurgery （43.8% , P 〈0. 05 ） although the survival rates of chimeric blastocysts were no different （91.4% vs 87.5% , P 〉0. 05 ）. Together, 1 ） Interspecific chimeric embryos （cattle and buffalo） reconstructed by ICM exchange are able to develop into the blastocyst; 2 ） Anticattle rabbit serum for isolating bovine ICM should be inactivated at 63.5℃ and performed in serum-free medium for 30 min ; 3 ） Immunosurgery is better in isolating ICM for chimeric embryo reconstruction compared with microsurgery.