Abstract
An efficient genome editing approach had been established to construct the stable transgenic cell lines in the domestic chicken (Gallus gallus domesticus) at present. Our objectives were to investigate gene function in the differentiation process of chicken embryonic stem cells (ESCs) into spermatogonial stem cells(SSCs). Three guides RNA (gRNAs) were designed to knockout the Stra8 gene, and knockout efficiency was evaluated in domestic chicken cells using cleavage activity of in vitro transcription of gRNA, Luciferase-SSA assay, T7 endonuclease I assay(T7E1) and TA clone sequence. In addition, the Cas9/gRNA plasmid was transfected into ESCs to confirm the function of Stra8. SSA assay results showed that luciferase activity of the vector expressing gRNA-1 and gRNA- 2 was higher than that of gRNA-3. TA clone sequencing showed that the knockdown efficiency was 25% (10/40) in DF-1 cells, the knockdown efficiency was 23% (9/40) in chicken ESCs. T7E1 assay indicated that there were cleavage activity for three individuals, and the knockdown efficiency was 12% (3/25). Cell morphology, qRT-PCR, immunostaining and FCS indicated that Cas9/gRNA not only resulted in the knockout of Stra8 gene, but also suggested that the generation of SSCs was blocked by the Stra8 gene knockdown in vitro. Taken together, our results indicate that the CRISPR/Cas9 system could mediate stable Stra8 gene knockdown in domestic chicken’s cells and inhibit ECSs differentiation into SSCs.
Highlights
Stra8 was a gene regulated by retinoic acid (RA), which was a specific expressed gene from mitosis to meiosis for mammalian germ cell
Stable knockout cell line has been produced in HEK293 cells and induced pluripotent cells produce stable knockdown cell lines, knockdown animals models had been achieved for mouse, rat and zebrafish by the microinjection method, the offspring was obtained successfully
Zhang et al [16] reported that the CRISPR/Cas9 system was suitable for editing gene in any cell types, the more study about this technique just focused on the mammalian cells
Summary
Stra (stimulated by retinoic acid gene 8) was a gene regulated by retinoic acid (RA), which was a specific expressed gene from mitosis to meiosis for mammalian germ cell. Previous investigator showed that Stra was expressed in embryonic and postnatal gonads [1]; it was expressed in cytoplasm and nuclei of germ cell[2]. When the cells were in different functional state, Stra could shuttle between the cytoplasm and the nucleus to play. Stra gene knockout and inhibit male germ cell differentiation
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