关键词: 绵羊, CRISPR/Cas9, 同源重组, sUMSCs, GFP, ROSA26

Abstract:

【Objective】The use of CRISPR/Cas9 system to establish a sheep tracing umbilical cord mesenchymal stem cell line lays the foundation for the clinical therapy and mechanism of MSCs. 【Method】Three single guide RNAs (sgRNA)were designed and synthesized for the ROSA26 locus of sheep by using the online tool ZiFiT Targeter Version 4.2, PCR was performed using the point mutation method with the px330 plasmid as a template. The PCR product was circularized after the plasmid DNA was removed by Dpn I, the sgRNA / Cas9 vector targeting sROSA26 was constructed after enzyme digestion and sequencing identification. The constructed plasmid contained Cas9 and sgRNA expression cassettes, which were driven by the U6 promoter. Because of the off-target effect of CRISPR/Cas9 system, the vector was transfected into sUMSCs, after extraction of its genome for PCR, T7E1 enzyme digestion. Then analyzing the gray levels of the bands to detect the vector editing activity. The sgRNA / Cas9 vector can cuts the double-stranded DNA at the target site. In order to knock in the reporter gene, the Donor vector needs to repair the sequence on the donor DNA by homologous recombination at the DNA break to insert target sequence. Based on the sgRNA sequence, the left and right homology arm amplification primers were designed and synthesized at the upstream and downstream of the sROSA26 locus. Using sheep's whole genome as a template for PCR amplification to obtain left and right homology arms. After recovery and purification, recombinant with pMD19-Simple to obtain left and right homologous recombinant plasmids. The left and right homology arm plasmids were ligated with the Donor expression vector DC-DON-SH02 ROSA26 to obtain the left arm recombinant targeting vector. The homologous recombination vector carrying green fluorescent protein (GFP) and puromycin resistance gene was constructed. The survival time of cells with different concentrations of puromycin was observed in well-growing sUMSCs to determine the optimal concentration and time of resistance screening. In sUMSCs, sgRNA / Cas9 vector and Donor vector were co-transfected by liposome method. Puromycin resistance screening was performed 48 hours after transfection. After the screening, cells need to be replaced with normal media and continue to expand. Observing the expression of GFP and extracting the positive cell genome , designing two pairs of upstream and downstream primers for ROSA26 site for PCR to detect its editing status. 【Result】(1) Three sgRNA primers were designed for the sheep ROSA26 locus. The expression vector px330-sgRNA1/2/3 were successfully constructed by point mutation method and the vectors were transfected into sheep umbilical cord mesenchymal stem cells. T7E1 enzyme assay results indicated that the editing efficiency of px330-sgRNA1 was the highest, about 20%. (2) Based on sgRNA1, the left and right homology arms of the targeting vector were obtained by PCR, and the recombinant vector(sROSA26-HA) of sheep ROSA26 locus was successfully obtained by a series of molecular biological methods; (3) px330-sgRNA1 and sROSA26-HA were co-transfected into sheep umbilical cord mesenchymal stem cells using Lipofectamine2000, and positive colonies could express green fluorescent protein were obtained after 1.5 μg·mL ^-1 puromycin selection for 15 days. PCR detection of one clone indicated that the targeting vector had been integrated into the genome. 【Conclusion】The exogenous GFP gene could successfully integrated in specific locus of sheep umbilical cord mesenchymal stem cells using the CRISPR/Cas9 system, and these cells can be used to trace the position and differentiated cell type of MSCs. This research laid a foundation for further clinical transformation of mesenchymal stem cells.

Key words: sheep, CRISPR/Cas9, homologous recombination, sheep umbilical cord mesenchymal stem cells, GFP, ROSA26