Derivation and Characterization of Embryonic Stem Cells Lines Derived from Transgenic Fischer 344 and Dark Agouti Rats

Abstract

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 b-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.

A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

“…The differences that exist between human and rat ESCs or between rat and mouse ESCs are yet to be fully elucidated. For example, we recently reported that rat ESCs maintained using the 2 inhibitor cocktail of PD0325901, a mitogen-activated protein kinase (MEK) inhibitor, and CHIR99021, a glycogen synthase kinase 3 (GSK3) inhibitor plus leukemia inhibitory factor (LIF), called two inhibitors (2i) below, express Cdx2 [6], a gene involved in trophoblast fate determination [7,8]. This finding is in sharp contrast to what is observed in mouse ESCs.…”

“…Rat ESC lines used here were derived from Dark Agouti (DA) and transgenic Fischer 344 (F344) rats. ESC derivation, ESC differentiation to embryoid bodies (EBs), and characterization of our DA and F344 ESCs was described previously [6]. In addition, 2i plus LIF genuine rat ESC pellets derived from DA rats were provided by Dr. Q. Ying (University of Southern California, Los Angeles, CA) [4].…”

“…Another difference is that rat ESCs cannot be maintained in medium containing only LIF when grown on inactivated mouse embryonic fibroblasts (MEFs), in contrast to mouse ESCs [9]. Finally, as reported for the mouse, strain differences may affect the quality of rat ESCs for producing germline transmission, or may affect the ability of the blastocyst to integrate ESCs and the efficiency of rat ESCs to contribute to the germline is lower than the mouse [3,4,6,10]. Therefore, distinct species differences exist between rat and mouse ESCs.…”

“…The gene list was provided to Qiagen and they manufactured the gene array. Next, we used this array to compare the gene expression of genuine rat ESCs produced in our laboratory [6] and from the laboratory of QY [4] using 2i medium and genuine ESCs produced using media containing four inhibitors (4i, the Rho-associated kinase inhibitor Y-27632; the MEK inhibitor PD0325901; the type 1 TGFb receptor inhibitor A-83-01; and the GSK inhibitor CHIR99021, called 4i below [10]. The 4i genuine rat ESCs were provided by the laboratory of MK and TO.…”

Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats

“…While a number of groups have derived ES cell lines and generated chimeras, it is clear that several lines then failed to demonstrate germline transmissibility [20,21]. Several factors, such as the genetic background, stemness, normality of karyotype, pathogen status of the ES cell line, as well as the genetic background of recipient embryos, affect the ES cells' ability to transmit their genetic material through the germline [13,14].…”