Effects Of TGF-?1 Gene Knockout Through Lentivirus-mediated CRISPR/Cas9 On Antler Cartilage Cells

Deer antler is a vastly special bone organ in mammals.It not only has the ability to periodically regenerate at an amazing growth rate,but also exhibits its powerful immune function through the scar-free healing of cutting wounds.So,it has become an ideal research model in various scientific research fields.The occurrence and regeneration of antler are regulated by many factors,and people still cannot fully map the regulation network of antler development.Transforming growth factor ?(TGF-?)is a multifunctional growth factor that has complex regulatory effects on cells.Studies have shown that TGF-? and its receptors are highly expressed at the top of deer antler,but their functional studies have rarely been reported.Therefore,exploring the vital role of TGF-? in the rapid growth stage of antler is the main purpose of this study.In this study,the bioinformatics software was used to predict,the nucleotide sequence and amino acid sequence similarity,the physicochemical properties of the gene-encoded protein,the post-translational modification site and the secondary structure,of the cloned sika deer TGF-?1 gene.And the relative expression intensity of TGF-?1 in different tissues of deer antler was analyzed by immunohistochemistry.The results showed that TGF-?1 was the highest in cartilage layer,followed by mesenchymal layer and skin layer.So as to explore the function of TGF-?1 in antler cartilage cells,we used the CRISPR/Cas9 gene-editing technology to knockout the TGF-?1 gene in antler cartilage cells.First,three gRNA oligonucleotide sequences targeting sika deer TGF-?1 sequence were designed,and after annealing,they were ligated with the linearized pBOBI vector,the positive clones were identified by sequencing.Then,the positive knockout vector was subjected to lentiviral packaging in 293 T cells by using lentiviral packaging system to enable the vector efficient entry into cartilage cells.Finally,the TGF-?1 knockout antler cartilage cells were screened by Western blot.After obtaining the TGF-?1 knockout cell lines of antler cartilage cells,the proliferation of gene knockout cartilage cells in vitro was detected by EdU assay,the migration of gene knockout cartilage cells was detected by cell scratching assay,the effect of gene knockout on the expression of TGF-? signaling pathway-related genes in cartilage cells was detected by PCR array assay and some of the results were verified by real-time PCR and Western blot.The results showed that the knockout of TGF-?1 inhibited the proliferation of antler cartilage cells in vitro and promoted the migration of cartilage cells,leading to the up-regulation of 11 related genes and the down-regulation of 9 related genes in TGF-? pathway.So,it is speculated that BMP4-mediated BMP signaling pathway may play a role of significant when the function of TGF-?1-mediated TGF-? pathway was lost.