Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells

Abstract

The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.

Keywords: 2i; chimaera; embryonic germ cell; embryonic stem cell; germline transmission; rat.

Fig. 1.. Rat pluripotent stem cell lines can be expanded using 2i+LIF as adherent cultures on MEFs and retain a stable karyotype.

(A) Phase contrast images of DA16 rat ES cells and WBY2 EG cells. Scale bars = 200 μm. (B) Schematic diagram of passaging technique. (C) Histogram showing chromosome number per cell in 50 metaphase spreads from line rat ES cell line DA16 at passage 13.

Fig. 2.. Transfection and clonal selection of rat pluripotent stem cells to generate stable transgenic lines.

Phase contrast and fluorescence images of (A) 16g2 clonal rat ES cells and (B) g2.5 clonal rat EG cells. Scale bars = 200 μm. Histogram showing chromosome number per cell in 50 metaphase spreads from (C) rat ES cell line 16g2 and (D) rat EG cell line g2.5 with accompanying metaphase spreads showing 42 chromosomes.

Fig. 3.. Genetically manipulated rat pluripotent stem cells contribute widely to chimaeras.

(A) Chimaeras from injection of 16g2 DA (agouti) rat ES cells into SD (albino) blastocysts. (B) Chimaeras from injection of g2.5 SD rat EG cells into SD blastocysts. Bright-field and fluorescence images of whole organs show contribution of (C) rat ES cells and (E) rat EG cells to a wide range of tissues. 3D projection of serial confocal sections through unfixed tissue squashes demonstrate the contribution of (D) rat ES cells and (F) rat EG cells to both tissue parenchyma and stromal cells. Chimaeras numbers 1 and 2 in (C) and (D) represent numbers 1 and 2 from (A).

Fig. 4.. Germ line transmission from high passage number, genetically manipulated rat pluripotent stem cells.

(A) Ear punches from wildtype littermate and agouti/fluorescent pups indicating germline transmission of 16g2. (B) Fluorescent pups indicating germline transmission of g2.5.

Fig. 5.. The DA strain is a suboptimal recipient for SD rat pluripotent cells.

(A) Bright-field and fluorescence images of two E11.5 chimaeric embryos and a litter mate control obtained from g2.5 injections into DA host blastocysts. (B) Bright-field and fluorescence images of two E13.5 chimaeric embryos and a litter mate control obtained from g2.5 injections into SD host blastocysts. (C) Summary of injections of unmodified SD rat EG cells into DA host blastocysts. (D) Low contribution adult chimaera from injection of WBY2 SD rat EG into DA blastocyst.