Biosafety Test Of VEGF Gene Editing Cashmere Goats

In recent years,Genome editing technology is the latest research technology for researchers to understand the function of specific genes and produce new gene-edited animals.With the completion of whole genome sequencing for more and more species,how to obtain gene-related function and application information from a large amount of data has become a new challenge for researchers.Gene-editing technology is a powerful research tool for achieving this important goal,and it has been named one of the top 10 important scientific advances in 2012 by the scientific community.In recent years,gene editing technology has developed rapidly,and zinc-finger nucleases(ZFN),TALE nucleases(Transcrition activator like effector nucleases(TALENs)and CRISPR/Cas9 system have emerged.CRISPR/Cas9 technology has the advantages of simplicity and efficiency.Since its inception,researchers have successively used this technology to produce genetically modified livestock such as cattle and sheep,which has shown a good application prospect in livestock breeding.With the rapid development of animal genetic editing technology,the industrialization of genetically edited animals has also shown a good application prospect.However,compared with genetically edited plants and microorganisms,the biosafety detection of genetically edited animals and their products is relatively lagging.In recent years,more and more attention has been paid to the biosafety of gene-editing animals,not only the safety of gene-editing animals and their products,but also the impact of gene-edited animals on the whole ecologicalenvironment.Therefore,the biosafety detection of gene-edited animals is particularly important,which is the only way for the industrialization of gene-edited animals.VEGF(vascular endothelial cell growth factor,VEGF)is a kind of vascular endothelial growth factor,can be expressed in the human and animal hair follicles,is one of the dermal papilla cells autocrine growth factor.It is an autocrine growth factor of dermal papilla cells which has the fuction of promoting hair growth.In this laboratory,CRISPR/Cas9 gene editing technology and somatic cell cloning technology were used to produce the fixed-point integrated cashmere goat of VEGF gene with Kap6.1 as the promoter.Compared with the wild type,this kind of cashmere goat showed good production characteristics and significantly improved the yield of cashmere.However,in the production of VEGF gene-edited cashmere goats,whether there are individual and environmental biosafety problems remains to be further studied under the premise of ensuring the increase in cashmere production of cashmere goats.Therefore,in this experiment,the VEGF gene-edited cashmere goats were expanded to obtain the offspring individuals.And the individual growth status,blood,tissues and organs and other physiological indicators of the offspring were detected,and gene drift detection was carried out in the relevant environment,so that to provide experimental basis for the future biosafety detection of gene editing animals.(1)Obtained the VEGF gene editing cashmere goat progenyIn this experiment,the VEGF gene-edited cashmere goat with ear number 1618 was subjected to superovulation,and after the embryo transfer,4 kids were obtained,and the ear numbers were 1812(?),1813(?),1817(?),1819(?),respectively.It was identified that No.1813,No.1817 and No.1819 were genetically edited positive.No.1819 was used for the expansion and growth index monitoring;No.1813(?)and No.1817(?)were slaughtered after weaning,taking the main tissues and organs and metabolites,and the cashmere goats in the same age and sex were used as controls,both individual safety and environmental safety of VEGF gene editing cashmeregoats were detected.(2)Biosafety test of VEGF gene-edited cashmere goatIn this study,VEGF gene expression was detected by q-PCR and Western Blot in two VEGF gene-edited cashmere goats.GAPDH gene and ?-tubulin gene were used as internal parameters,and the results were compared with those of cashmere goats in the control group.The results showed that the expressions of mRNA in the skin of No.1813 and No.1817 cashmere goats were 2.07 and 2.24 times than that of those in the control group,respectively.There was no significant difference between the expressions of mRNA in the heart,liver,spleen,lung and kidney tissues and those in the control group.Western Blot results were consistent with qPCR results.After that,blood including liver function and kidney function and other blood routine,biochemical detection,as well as the metabolism of fatty acids and amino acids in various tissues and organs were carried out.The results showed that there was no significant difference between the indexes of VEGF gene editing cashmere goats and the control group.Subsequently,16 S rRNA was detected in intestinal microorganisms of VEGF gene-edited cashmere goats and controls.The statistical data of intestinal microorganisms in phylum,class,order,family and genus were obtained.The results showed that there was no significant difference in intestinal microbial composition and abundance between VEGF gene-edited cashmere goats and controls.We continued to track the individual growth status of VEGF gene-edited cashmere goats,including body length,height,weight and chest circumference at various stages,and drew the growth curve.The results showed no significant difference from the control group.(3)Environmental safety detection of VEGF gene-edited cashmere goatsIn order to detect environmental safety,we performed intestinal microbial drift and microbial drift in surrounding soils in terms of gene drift.As a result,no exogenous genes were detected in the genomes of intestinal microorganisms and soil microorganisms.The above experimental results showed that the exogenous genes of VEGFgene-edited cashmere goats with Kap6.1 as the promoter had specific expression in skin tissue.There was no change in the expression level in other tissues and organs,which had no obvious effect on individual growth and development,and no gene drift.The above detection provides experimental basis for further research on the biosafety of gene-edited animals in the future.The results show that VEGF gene-edited cashmere goat has a good application prospect in cashmere goat breeding.