Targeted Knockout And Functon Of Fam49b Gene In Human Cell Lines

Genome editing is an important approach in modern biological research and is widely used to study gene function by manipulating the genetic sequence. In recent years, three new genome editing technologies ZFN, TALEN, and CRISPR/Cas9 have became increasingly prevalent. With advantages of convenient operation and high targeting efficiency, CRISPR/Cas9 technology has rapidly became a new generation of genome editing technology. Research on Fam49b gene has rarely been reported. Our previous studies showed that Fam49b may participate in Xenopus neural crest development by regulating cell proliferation and/or apoptosis. The objective of this study is to establish a Fam49b knockout human cell line with CRISPR/Cas9 technology and analyze its biological characteristics.The main methods and results of this study are as follows:1. RT-PCR and Western blot assays confirmed the expression of Fam49b gene in three human cell lines HEK293T, U87, and U251, and immunocytochemistry (ICC) results showed that Fam49b protein is widely expressed in the cytoplasm and nucleus.2. Two targeting sites T1 and T2 of human Fam49b gene were designed, then the CRISPR/Cas9 targeting vectors were successfully constructed, and finally their targeting efficiencies in HEK293T cells were detected as 45% and 32%, respectively.3.21 HEK293T monoclonal cell strains were successfully established through limiting dilution, in which a Fam49bl isoform knockout cell strain--No.7 monoclonal cell strain was identified by PCR-RFLP and Western blot, and then the Fam49b allele sequences of No.7 cell strain were confirmed by sequencing.4. Through further research on Fam49b transcripts and proteins of No.7 cell strain, a new isoform of human Fam49b gene--Fam49b2 was discovered and the alternative splicing mode of human Fam49b gene was analyzed by combining with the bioinformatics information.5. Flow cytometry and CCK-8 assay were used to study the biological characteristics of No.7 cell strain, which indicates that loss of Fam49b1 may inhibit cell proliferation.6. CRISPR/Cas9 technology was applied to U251 cell line and a U251 cell line with high mutation rate of Fam49b gene was successfully established through fluorescence activated cell sorting (FACS).This study is the first attempt to precisely modify human Fam49b gene using CRISPR/Cas9 technology. We established a Fam49bl knockout HEK293T cell line, and found that proliferation ability of this cell line decreased. Not only does this study contribute to the understanding of Fam49b functions in human cells, but also provides an effective human cell model for intensively studying roles and mechanisms of Fam49b gene. At the same time, we discovered a new isoform of human Fam49b gene, which improved our understanding of the forms of human Fam49b protein and also helped us fully understand Fam49 gene family. In addition, the application of CRISPR/Cas9 technology to U251 cell in this study helps in subsequently screening gene knockout cell lines, and provides a valuable reference for the use of CRISPR/Cas9 technology in low transfection efficiency cell lines.