小鼠胚胎生殖细胞系的建立及其印记状态

Establishment of a mouse embryonic germ cell line and preliminary study of the expression of imprinted genes

目的成功建立小鼠胚胎生殖细胞(EGCs)系,并初步分析小鼠胚胎生殖细胞的印记状态。方法建立交配后12.5d(12.5dpc)原始生殖细胞(PGCs)来源的小鼠EGCs,通过碱性磷酸酶(AKP)染色、免疫荧光细胞化学、体内分化及体外分化等方法检测EGCs的多能性,并以小鼠胚胎干细胞(ESCs)为对照,应用Real-time PCR检测EGCs中与发育相关的Ins2、Lgf2、H19、Lgf2r等11个父源与母源印记基因的表达情况。结果成功建立小鼠EG细胞系,EGCs克隆AKP染色显示有高水平的AKP活性,免疫荧光细胞化学方法显示克隆表达小鼠ESCs多能性标记物Oct4及细胞表面标记SSEA-1。核型分析检测显示,小鼠EGCs为正常的40条染色体,体内可分化出3个胚层来源的组织,说明小鼠EGCs具有多能性;Real-time PCR结果显示EGCs的印记基因表达量显著高于ESCs。结论12.5dpc PGC来源的EGCs的印记基因处于擦除状态。

Objective To establish a mouse embryonic germ cell （EGCs） line and to detect the expression of imprinted genes in order to provide basic information for further study and application of embryonic germ cells. Methods EGCs were isolate from primordial germ cells collected from the genital ridge of 12.5 days postcoitum （dpe）. The pluripotent characteristics of the established EGCs were detected by alkaline phophatase （ AKP ） staining, immunofluoreseent detection of mouse embryonic stem cells （ESCs） surface antigens, and cell differentiations in vivo. The expressions of several patrilineal and matrilineal imprinted genes, such as Ins2, Lgf2, H19, Lgf2r and so forth, were also detected by quantitative reverse transeriptase-polymerase chain reaction in both EGCs and ESCs. Results The EGCs showed positive for alkaline phosphatase. The pluripoteney marker Oct4 and the cell surface marker SSEA-1 were also shown in EGCs cells. Karyotype analysis indicted that EGCs had normal 40 chromosomes, and differentiated into the tissues presenting three germinal layers derivations in vivo, suggesting that embryonic germ cells had pluripotent characteristics. Real-time PCR showed that the expression levels of imprinted genes in EGCs were significantly highter compared with those in ESCs. Conclusion The gcnomic imprinting memories in EGCs generated from primordial germ cells which collected from the genital ridge of 12.5 dpc are completely erased.