Establishment Of CRISPR-Cas9 Mediated CD13-Knockout Cell Lines Of Porcine Intestinal Epithelial Cells

CD13 is a zinc ion-dependent protease.CD13 is distributed on the surface of various tissue cells,has the function of hydrolyzing neutral amino groups,participates in signal transduction and immune regulation,promotes tumor migration,and also acts as an adhesion-binding receptor for some pathogenic microorganisms.It has been widely studied in recent years.IPEC-J2 and IPI-2I are two types of porcine intestinal epithelial cell lines that are widely used to study interactions with pathogenic microorganisms.In this study,two small guide RNAs,g3 and g5,were designed corresponding to the second exon region of CD13 gene.The g3 and g5 g RNAs were cloned into the p X330-GFP and p X330-RFP vector backbones,respectively.The constructed vectors were transfected into DH5 alpha competent cells,coated with ampicillin-containing LB plate for growth,and single colonies were selected for sequencing.Choose the right colony for enlarged culture and extract endotoxin-free plasmid to provide experimental materials for subsequent experiments.PX330-g3-GFP and p X330-g5-RFP were simultaneously transfected into IPEC-J2 cells by electrotransfection.After 48 hours of transfection,red-green double-fluorescence screening was performed by flow cytometry and some positive cells were separated into 96-well cell culture plates.One cell in each well was cultured as monoclonal cells.After monoclonal cells were cultured,PCR amplification,electrophoresis and sequencing were performed to obtain CD13 gene homozygous knockout positive cell lines.Western blot was used to detect the expression level of homozygous fragment knockout cells.CCK-8 was used to detect and plot the growth curve of CD13 knockout cells.Scratch healing test and Transwell test were used to the migration ability of knockout cells.The results showed that 20 monoclonal cells were obtained and amplified by PCR and some cloned PCR products were sequenced.Seven clones were found to have deletion of fragments,three clones had deletion of biallelic heterozygous fragment,and the CD13 protein of biallelic homozygous fragments knockout cells was almost not expressed,suggesting that we successfully constructed CD13 knockout IPEC-J2 cell lines;CCK-8 proliferation test results showed that there was no significant difference in the proliferation rate between knockout cells and wild-type cells;scratch healing test showed that there was no significant difference in the migration rate between the two cells;Transwell test showed that there was no significant difference in the migration rate between the two cells.Using the same method,the two plasmids were transfected into IPI-2I cells at the same time.The results of PCR and sequencing of the obtained 310 cell clones showed that a total of 48 clones had fragment deletions,32 clones with biallelic fragment deletions,and 23 with biallelic homozygous fragments deletions.Western blot showed that CD13 protein was almost not expressed in 272 bp biallelic heterozygous fragment deletions cells,suggesting that CD13 gene was successfully knocked out in IPI-2I cells.CCK-8 was used to detect proliferation,and the results showed that there was no significant difference in proliferation rate compared with wild-type cells;scratch test showed that there was no significant difference in migration rate between the two types of cells;Compared with wild-type cells,the migration rate of CD13 knockout IPI-2I cells was not significantly different by Transwell test.In this experiment,two CD13 knockout cell lines of porcine small intestinal epithelial cells were made and their partial abilities were studied.It provides experimental materials for further study of the interaction between CD13 and pathogenic microorganisms on porcine intestinal epithelium,and other functions of CD13 gene.