具生殖系嵌合能力的C57BL/6J小鼠胚胎干细胞系的建立

Establishment of a Germ-line Transmission Using C57BL/6J Embryonic Stem Cell Lines

目的:建立能实现种系传递的C57BL/6J小鼠胚胎干细胞系。方法:从C57BL/6J小鼠3.5d的囊胚中分离培养内细胞团。经体外适宜培养建系后,将C57BL/6J胚胎干细胞(ES)注入ICR小鼠受体囊胚腔,制备嵌合体小鼠。结果:成功地建立了3个C57BL/6J小鼠胚胎干细胞系,该C57BL/6JES细胞呈集落状生长,正常稳定的核型率>80%,具高水平的磷酸酶活性,并表达ES细胞特殊性细胞表面标记SSEA-1,不表达SSEA-3和SSEA-4;体内外的分化实验也证实mC57ES1具向三胚层组织分化的能力。经显微注入ICR小鼠囊胚腔中,产生4只嵌合体小鼠,经证实其中1只为种系嵌合小鼠。结论:建立了能实现种系传递的C57BL/6J小鼠胚胎干细胞系,该系的ES细胞可用于今后制备转基因动物和基因敲除动物。

Objective: To establish C57BL/6J embryonic stem(ES)cell lines with potential germ-line contribution. Methods: ES cells were isolated from C57BL/6J mice blastocyst inner cell mass, and cultured for 15 passages, then injected into blastococels of ICR mice blastocysts to create chimeric mice. Results: Three ES cell lines (mC57ES1, mC57ES3, mC57ES7) derived from C57BL/6J mice blastocyst inner cell mass were established. They were characteristic of undifferentiated state, normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-1” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transfered into ICR mice blastocysts, 4 chimeric mice were obtained, one male of whom exhibited successful germ-line transmission. Conclussion: We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be applied to generate transgenic and gene knock-out mice.