ES cell lines derived from Stra8-EGFP/Sycp3-DsRed mice…

ES cell lines derived from double transgenic (dTg) Stra8-EGFP and Sycp3-DsRed mouse line (Smorag et al., 2012) were generated to be used in in vitro spermatogenesis experiments. These transgenic mice provide the premeiotic Stra8-EGFP and meiotic Sycp3-DsRed constructs (Fig. 50).

Fig. 50: Schematic representation of promoter constructs of double transgenic Stra8-EGFP and Sycp3-DsRed mice. kb: kilo base of promoter region. Modified, Nolte et al., 2010.

3.4.2.2.1 Generation of Stra8-EGFP/Sycp3-DsRed ES cell lines

The dTg Stra8-EGFP and Sycp3-DsRed mouse line (Smorag et al., 2012) was used for the generation of Stra8-EGFP/Sycp3-DsRed ES cell lines. In brief, blastocysts were isolated from gestating mice at stage 3.5 days post coitum (dpc) and cultured on MEF feeder layer in KSOM medium at 37°C and 5% CO₂. After outgrowth of the inner cell mass of the blastocysts, it was disaggregated and seeded on fresh MEF feeder layer. During further culture in ESC medium cells grew in colonies and revealed a mouse ES cell like morphology (2.2.4.10).

3.4.2.2.2 Characterization of Stra8-EGFP/Sycp3-DsRed-2 ES cells

While several ES cell lines could be generated from dTg mice, Stra8-EGFP/Sycp3-DsRed-2 cells were chosen for further characterization and experimental procedure (Fig. 51 A). Its pluripotent character was proven by the detection of OCT4 protein expression using western blot analysis. ES-RI was used as a positive control, whereas the negative control CRL-2691, a male human fibroblast cell line, did not reveal any expression of the pluripotency marker (Fig. 51 B). Furthermore, a male genotype of Stra8-EGFP/Sycp3-DsRed-2 was required for its intended use in in vitro spermatogenesis experiments. Aiming to check for its male genotype, Stra8-EGFP/Sycp3-DsRed-2 cells were examined for the presence of the androgen receptor (AR) and sex determining region of chromosome Y (SRY). Genomic DNA of a male wild type mouse was used as a positive control and revealed detection of both genes analyzed, whereas Sry could not be detected in female wild type mouse used as a negative control. Compared to the controls Stra8-EGFP/Sycp3-DsRed-2 demonstrated a male genotype (Fig. 51 C).

Finally, genotyping PCR confirmed that Stra8-EGFP/Sycp3-DsRed-2 cells indeed derived from dTg animals and contained the transgenes Stra8-EGFP and Sycp3-DsRed (Fig. 51 D).

Analysis was performed according to Smorag et al. (2012) and genomic DNA of a wild type ES cell line was used for control.

Fig. 51: Characterization of the generated ES cell line Stra8-EGFP/Sycp3-DsRed-2. (A) Representative brightfield image of the generated ES cell line Stra8-EGFP/Sycp3-DsRed-2. Scale bar: 200 µm. (B) Western blot analysis revealed the expression of the pluripotency factor OCT4 in Stra8-EGFP/Sycp3-DsRed-2 cells. Wild type ES cell line ES-RI was used as positive control, human fibroblast cell line CRL-2691 as negative control.

Detection of α-tubulin served as loading control. Molecular weights of proteins are indicated. kDa: kilo Dalton.

(C) Stra8-EGFP/Sycp3-DsRed-2 cells possessed a male genotype by expressing Ar and Sry. Genomic DNA of male and female mice served as controls. Ar: Androgen receptor; Sry: sex determining region of chromosome Y;

NC: negative control with H2O. (D) Genotyping PCR proved the derivation of Stra8-EGFP/Sycp3-DsRed-2 cells from homozygous double transgenic mice. 2Trg: genomic DNA of double transgenic mice; WT: wild type genomic DNA; NC: negative control with H2O; trg: transgene. Stra8 trg: genotyping PCR for Stra8 transgene; SYCP trg:

genotyping PCR for Sycp3 transgene. PCR fragment sizes are indicated within the figures C and D.

3.4.2.2.3 Enrichment of EGFP positive cells by FACSorting

According to the strategy for the derivation of male germ cells from stem cells, an enrichment of EGFP positive (EGFP⁺) cells resembles a spermatogonial population (Nayernia et al., 2006; Nolte et al., 2010), which requires a retinoic acid (RA) inducible EGFP expression in the used cell line. For validation of RA inducible EGFP expression, Stra8-EGFP/Sycp3-DsRed-2 cells were cultured in the presence of RA (10^-6 M) for three days and checked for EGFP expression by fluorescence microscopy (Fig. 52 I). In comparison to non-RA-induced cells, application of RA activated the Stra8 promoter and

Stra8-EGFP/Sycp3-DsRed-2 cells. For enrichment of EGFP⁺ cells Stra8-EGFP/Sycp3-DsRed-2 cells were induced with RA for three days and EGFP⁺ cells were collected by FACSorting (Fig. 52 II). The fraction of EGFP⁺ cells could be raised by

~32% resulting in a population of ~82% EGFP⁺ cells (Fig. 52 IIA). These cells were cultured on MEF feeder layer in ESC medium containing RA in order to further increase the EGFP⁺ cell population, which should be obtained by a second FACSorting. Here the initial EGFP⁺ cell population revealed ~56% EGFP⁺ cells, which could be enriched in a fraction consisting of ~87% EGFP⁺ cells by repeated FACSorting (Fig. 52 IIB). These cells were further cultured on MEF feeder layer in ESC medium containing RA. ES cells derived from a murine C57BL/W background were used as control in FACSorting analyses and Stra8-EGFP/Sycp3-DsRed-2 cells before and after FACSortings were cyropreserved in a sufficient number of stocks for subsequent analyses.

Fig. 52: RA inducible EGFP expression used for FACSortings of Stra8-EGFP/Sycp3-DsRed-2 ES cells.

(I) Stra8-EGFP/Sycp3-DsRed-2 colonies revealed strong EGFP expression after three days of RA induction.

-: without RA induction; +: with 10^-6 M RA induction for three days; RA: Retinoic Acid. Representative scale bars are indicated within overlay images. Scale bars: 20 µm. (II) EGFP positive cells were enriched by two FACSortings (A + B) of RA induced Stra8-EGFP/Sycp3-DsRed-2 cells. Percentages of EGFP positive cells