Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506, USA.
Stem Cells Dev. 2012 Jun 10;21(9):1571-86. doi: 10.1089/scd.2011.0370. Epub 2011 Nov 22.
Rat embryonic stem cell (ESC) lines are not widely available, and there are only 2 lines available for distribution. Here, ESC lines were derived and characterized from Fischer 344 (F344) rats that express marker transgenes either β-galactosidase or human placental alkaline phosphatase (AP), nontransgenic F344 rats, and from Dark Agouti (DA) rats. The ESC lines were maintained in an undifferentiated state as characterized by colony morphology, expression of Oct4, Nanog, Sox-2, Cdx2, and Stella, staining for AP, and stage-specific embryonic antigen-1. Pluripotency was demonstrated in vitro by differentiation to embryoid bodies, followed by embryonic monsters. The Cdx2 expression by ESCs was unexpected and was confirmed via reverse transcriptase-polymerase chain reaction, immunocytochemistry. Pluripotency of ESCs was demonstrated in vivo by production of teratoma after an injection into F344 nontransgenic rats, and by an injection of male DA ESCs into F344 or Sprague-Dawley rat blastocysts and the generation of chimeric rats and germline contribution. ESCs from both F344 and DA contributed to chimeric rats, and one DA ESC line was proved to be germline competent. ESC sublines were created by transfection with a plasmid expressing enhanced green fluorescent protein (eGFP) under the control of a beta actin promoter and cytomegalovirus enhancer (pCX-eGFP) or by transfection with a plasmid expressing GFP under the control of a 3.1 kb portion of the rat Oct4 promoter (pN1-Oct4-GFP). In pN1-Oct4-GFP sublines, GFP gene expression and fluorescence were shown to be correlated with endogenous Oct4 gene expression. Therefore, these new ESC lines may be useful for tissue engineering and transplantation studies or for optimizing culture conditions required for self-renewal and differentiation of rat ESCs. While they made chimeric rats, further work is needed to confirm whether the transgenic F344 rat ESCs described here are germline-competent ESCs.
大鼠胚胎干细胞（ESC）系并不广泛可用，并且仅有 2 种可供分发。在此，从表达β-半乳糖苷酶或人胎盘碱性磷酸酶（AP）标记转基因的 Fischer 344（F344）大鼠、非转基因 F344 大鼠和 Dark Agouti（DA）大鼠中衍生和表征了 ESC 系。ESC 系通过集落形态、Oct4、Nanog、Sox-2、Cdx2 和 Stella 的表达、AP 染色以及阶段特异性胚胎抗原-1 的表达来表征为未分化状态。通过向 ESC 中导入胚胎体，随后向 ESC 中导入胚胎体，在体外证明了多能性。通过逆转录聚合酶链反应、免疫细胞化学证实了 ESC 中 Cdx2 的表达。通过将 ESC 注射到 F344 非转基因大鼠中产生畸胎瘤以及将雄性 DA ESC 注射到 F344 或 Sprague-Dawley 大鼠胚泡中并生成嵌合大鼠和种系贡献，在体内证明了 ESC 的多能性。来自 F344 和 DA 的 ESC 均有助于生成嵌合大鼠，并且一个 DA ESC 系被证明具有种系能力。通过转染表达在β肌动蛋白启动子和巨细胞病毒增强子（pCX-eGFP）控制下的增强型绿色荧光蛋白（eGFP）的质粒或通过转染表达在大鼠 Oct4 启动子的 3.1kb 部分控制下的 GFP 的质粒（pN1-Oct4-GFP）创建了 ESC 亚系。在 pN1-Oct4-GFP 亚系中，GFP 基因表达和荧光与内源性 Oct4 基因表达相关。因此，这些新的 ESC 系可能对组织工程和移植研究有用，或者对大鼠 ESC 自我更新和分化所需的培养条件进行优化。虽然它们生成了嵌合大鼠，但需要进一步的工作来确认这里描述的转基因 F344 大鼠 ESC 是否是种系能力的 ESC。
