| Objective: Growth Hormone(GH)plays an important role in physiological processes such as mammalian growth,development,and metabolism.It is exercised in animals by combining with growth hormone receptors(GHR)on the surface of target cell membranes.biological functions.If GHR is mutated,GH will lose its biological effects and cause adverse consequences,such as human Laron syndrome.Bama pigs have the characteristics of small size,long life span,short pregnancy cycle,large litter size,strong anti-infection and disease resistance.In addition,Bama pigs are very similar to humans in physiological and pathological medical characteristics.Disease model animals have extremely high research value in biomedical research.This study intends to prepare GHR knockout Bama pig cells by CRISPR/Cas9 technology,in the hopethat in the future,somatic cloning technology can be used to produce more "mini" GHR knockout Bama pigs.The more "mini" Bama pigs will be more conducive to experimental operations and can reduce the cost of feeding and management,and can be used as an animal model of Laron syndrome to lay the foundation for medical research.Methods: 1.The primary kidney fibroblasts of male and female Bama pigs were obtained by tissue block attachment method and trypsin digestion method.2.The two sets of sg RNA were selected and designed through the sg RNA design tool(http://crispr.mit.edu/),the CRISPR/Cas9 editing plasmid which targeted the first exon of the GHR was constructed through annealing,primers and plasmid ligation,and then transformation,picking and expansion.3.The liposome transfection and screening protocol was optimized,and the editing efficiency of the targeted target sites of the two groups of plasmids was compared by direct sequencing of two sets of CRISPR/Cas9 plasmids.4.The plasmids with higher transfection efficiency was selected to transfect male and female kidney fibroblasts,the mutant monoclonal cell lines were selected and isolated,and all mutant genotypes were obtained by sequencing.5.Monoclonal cell lines were tested for plasmid vector integration and off-target analysis.Results: 1.Several tubes of Bama pig fibroblasts were successfully collected and frozen by tissue block attachment method and digestion method.2.The CRISPR/Cas9 plasmids were successfully constructed: GHR-Cas9-1 and GHR-Cas9-1.After the plasmid was verified by double digestion,the band sizewas consistent with the original plasmid length,and the direct sequencing results were completely consistent with the sg RNA sequence.3.The optimization results of liposome transfection scheme: plasmid transfection amount 1 ?g,Lipofectamine3000 amount 3.75 ?L,puromycin concentration used for screening 1.5 ?g/m L: direct sequencing results confirmed that GHR-Cas9-1plasmid had higher transfection efficiency.4.Twelve male monoclonal cell lines and 10 female monoclonal cell lines were isolated through transfection and screening.After sequencing analysis,it was found that 21 out of 22 monoclonal cell lines had double allele mutations,and the gene editing efficiency was95.45%.5.The Cas9 plasmid vector integration test results as follows: the male monoclonal cell line # 1,# 4,# 6,# 7,# 8,# 10,and # 12 did not have Cas9 plasmid integration;the female single cloned cell lines # 1,# 5,# 6,# 7,# 8,and# 9 did not have Cas9 plasmid integration,the plasmid vector integration rate was 40.9%;The off-target prediction analysis of male monoclonal cell lines # 1and # 4 and females # 1 and # 5 revealed that no off-target occurred in the four cell lines.Conclusion: The 21 monoclonal cell lines with GHR gene mutation were successfully obtained,and each mutation genotype was successfully analyzed.The integration test of plasmid puromycin site showed that the integration rate of plasmid vector was 40.9%,and the four selected cell lines had no off-target situation. |
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