五指山近交猪胚胎生殖细胞(EG)嵌合体在我国移植产仔  

The First Chimeric Piglets was Born from Blastocyst Injection of Primordial Germ Cells of Wuzhishan Inbred Pig in China

作　　者：冯书堂[1] 丛笑倩[2] 刘立新[1] 李秀兰[3] 董晓[1] 包林平[2] 张莉[1] 陈红平[1] 牟玉莲[1] 况玲[1] 张青峰[1] 姚錱[2] 

机构地区：[1]中国农业科学院畜牧研究所 [2]中国科学院生化细胞研究所 [3]中国科学院生化细胞研究所,上海200031

出　　处：《畜牧兽医学报》2005年第3期311-312,共2页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金97重大项目(39993420)

摘　　要：采集25～28日龄的五指山小型猪(WZSP)胚胎原始生殖嵴细胞(PGCs),用于WZSP生殖细胞(EG)建系培养技术研究.研究发现:以DMEM含15%胎牛血清、0.1 mol/L巯基乙醇等为基础培养液,分别添加生长因子20 ng/mL LIF、40 ng/mL hSCF、20 ng/mL hbFGF,以STO为饲养层,进行EG培养建系,WZSP 3 d可出现克隆,其克隆具有出现早,生长数量多,增殖快,成熟亦早,传代率高的特点.培养7～10 d需传代或冷冻保存.EG类细胞冷冻、解冻后尚可复苏,并传至11代.若基础培养液不添加生长因子,培养6～10 d后亦可出现EG克隆细胞株,但其克隆具有出现晚、数量少、增殖慢等特点.研究中还发现25～26日龄的WZSP胚胎的原始生殖嵴小、不易观察、PGCs分离较困难;27～28日龄的WZSP胚胎的原始生殖嵴体积大、易观察、便于分离并可获得多量的PGCs.同时对PGCs进行了形态学、体外分化、AKP染色鉴定等研究.2002年9-12月,在中国农业科学院畜牧研究所五指山猪保种场先后从22头超排处理的大白猪供体中,于配种后(当天为0 d)5.5～7 d实施手术法获得252枚可用胚.将123枚胚胎显微注射WZSP的EG细胞,每枚注射5～15个细胞,与未注射的103枚混合分别移植给9头受体,平均每头受体移植胚胎25.11±5.11枚、含注射EG细胞胚胎13.67±3.57枚,其中7头分别于移植15～22 d返情,1头妊娠,1头待观察.其中2002年9月17日移植30枚胚胎的133号受体,于2003年元月8日产仔8头,其中6头为白色毛仔猪,2头表现为皮肤色素嵌合.Porcine primordial germ cells(PGCs) were isolated from day 25 to 28 genital ridges of fetuses from inbred Wuzhishan miniature pig (WZSP).To support undifferentiated growth, cell lines were derived and stable maintained on STO,which was used for in vitro culture,clone,isolation and passage of embryonic germ cell(PEG).The culture basic medium (CBM) was DMEM with 15% FCS+0.1mmol/L 1-mer-captoethanol+2 mmol/L glutamine and so on.The result was shown that if CBM with growth-stimulating factors such as SCF,bFGF and LIF,the EG’s clone was coming out more early than CBM alone and also with fast growth rate and undifferentiate characteristics than CBM clone.The PGCs of fetuses between day 27~28 of pregnancy (estrus=day 0) was easy and more suitable for collection.PEG’s cell could be survival in vitro culture after frozen and thawing and passaged over F_(11). The experiment of making chimeric pig by PGCs was carried out from Sept.to Dec.2002 at experimental farm on Institute of Animal Science,CAAS.Total 252 embryos at blastocyst stages was collected from 22 donors of Large White pig at day 5.5 to 7 of breeding(estrus=day 0).123 of 226 embryos were used for PGCs injection,each embryo was injected 5 to 15 cells of PGCs on average.226 embryos were transferred to 9 recipients,7 of them were return to estrus after transfer at day 15 to 22,one of them remained pregnancy,and the other needs to be future tested.8 piglets were delieved at January 8,from the recipient which was transferred 30 embryos (18 embryo injected and 12 embryo no-injection).2 of 8 piglets were showing coat colour skin,that is called a piglet’s chimeras.The future evaluation of molecular genetic testing is needed to do.

关 键 词：移植 胚胎 细胞 注射 受体 观察 生长因子 产仔 日龄 近交 

分 类 号：S828[农业科学—畜牧学] R329[农业科学—畜牧兽医]