Construction Of FGF5 Knockout Liaoning Cashmere Goat Fetus Fibroblast Cell Line

Fibroblast growth factors 5(FGF5)is one of the most important growth factors that affects the periodically growth of hair follicle.In this study the Liaoning cashmere goat fetal fibroblasts of FGF5 knockout were built by CRISPR/Cas9 systerm,to establish the foundation for study its biological functions further.On the basis of the coding region sequence information of cashmere goat FGF5 gene(KC236981.1),the primers were designed,and the complete CDs(Coding sequence)region was amplified,in which the spliceosome FGF5 s was not found.The results showed that the sequences length of FGF5 was 813 bp encoding one opening reading fram with 270 amino acid residues.The molecular weight of FGF5 was 29.55 kDa,and the theoretical pI was 10.59.FGF5 was a hydrophilic protein and has signal peptide,which makes it belonged to secreted protein.The amino acids homology between cattle reached 97.2%.The fibroblast cell were isolated from Liaoning cashmere goat fetuses by issue explants adherent method.In order to select the donor with strong growth performance and normal karyotype,the sex identification,growth curve and karyotype analysisit were carried out.The results showed that the Liaoning cashmere goat fetus fibroblasts in vitro culture system was successfully established.Cell line S1 was female while S2 was male.They were in good condition,with normal karyotype(30 pairs of chromosomes).Five predicted sgRNA targeted to first exon of FGF5 were designed by biology software,and 5 CRISPR/Cas9 recombinant vectors(PX459-FGF5-l,PX459-FGF5-2,PX459-FGF5-3,PX459-FGF5-4 and PX459-FGF5-5.)were constructed,which were identified by sequencing.The fetus fibroblasts were transfected with recombinant vectors by lipidosome 3000 respectively.The deletion efficiency was examined by T7 EI digestion,and the cells with higher deletion efficiency were passaged to single cell.After expanding and propagating,the genomic DNA were extracted,which were used to PCR,off-target and sequencing.The results showed that two vectors had the ability of specific cutting and mutation on the present site.In the 60 monoclonal cells chosen randomly,23 lines were FGF5 knockout lines(Indel and Substitution)and the mutation rate was 38.3%.In conclusion,the nine transgenic cells were suitable as the donor cell for somatic nuclear transfer,which will lay the foundation for the cultivation of new kinds of Liaoning cashmere goats.