Establishment And Experimental Study Of FAM76B-Luciferase-KI HEK293 Cell Line Based On CRISPR/Cas9 Targeted Genome Editing Technology

FAM76B(Family with sequence similarity 76B,FAM76B)is located on human chromosome I 1 q21.The overall length of the FAM76B gene is 21,468 bp,and the cDNA is 1,020 bp.It has 10 exons,encoding 339 amino acids,which molecular weight is about 38.7 KD.The mature FAM76B protein is located in the nucleus and distributed in spots.By comparing the amino acid sequence of FAM76B between human and mouse,it was found that the homology of FAM76B gene was as high as 98%between the two species.Therefore,FAM76B protein was highly conserved.At present,there are few studies on the function of FAM76B gene.According to the previous study on FAM76B molecule in our laboratory,the results showed that FAM76B gene was widely expressed in various tissues of mice.The expression level of brain was the highest,followed by liver and spleen.Knockout of FAM76B gene led to abnormal lipid metabolism in mouse liver,but its mechanism was still unclear.Therefore,using CRISPR/Cas9 genome editing technology,we established a new cell line named FAM76B-Luciferase-KI HEK293 cell line.This cell line was constructed by inserting luciferase reporter gene into the downstream of FAM76B gene.In this cell line,luciferase expression level was used to reflect the expression level of FAM76B gene,sensitively and accurately.And this reporting system can be used to detect the FAM76B expression level quantitatively.This new cell line can be used as a tool to study the expression regulatory mechanism of FAM76B.And,it can be applied to screen the transcription factors and small molecule drugs affection the expression of FAM76B gene,which is beneficial to explore the function of FAM76B gene.The research content of this project includes the following aspects:1.FAM76B sgRNA design,activity detection and the target vector construction:First,the sequence of human FAM76B genome was obtained,and the region located in the downstream of the termination codon of FAM76B gene was selected as the target region of sgRNA.Then,three potential sgRNA binding sites with high specificity were screened in the target region by sequence analysis.The sgRNA with high biological activity was obtained with T7 El assay.Then the vector co-expressing sgRNA with high biological activity and Cas9 was constructed,which can be used for the establishment of subsequent cell line.2.Construction of a donor vector targeting FAM76B gene:Using molecular biology technology,a donor vector was designed and constructed successfully,which includes upstream and downstream homologous arm of the target region of FAM76B gene,luciferase reporter gene,eGFP,neomycin,mCherry and TK gene.In the donor vector,luciferase,eGFP and neomycin were located between the upstream homologous arm and the downstream homologous arm in which eGFP and neomycin were positive screening elements,and mCherry and TK genes were located on the outside of the downstream homologous arm,which were used for negative screening elements.Positive and negative screening expression elements in the target vector can directly distinguish the cells with homologous recombination from that with random integration through the expression of eGFP or mCherry,at the same time,the cells with homologous recombination can be enriched by the screening of neomycin and TK gene,which can efficiently increase the proportion of the positive cells with homologous recombination.3.Establishment and identification of FAM76B-Luciferase-KI HEK293 cell line:First,the target vector and the donor vector were co-transfected into HEK293 cells,and the obtained cells were screened by G418 and GCV.After drug screening,the cells were cloned by limited dilution method,and the cloned cells were identified by PCR and sequencing.Finally,a single stable cell line was obtained,named FAM76B-Luciferase-K1 HEK293 cell line,which luciferase was specifically inserted at the downstream of FAM76B gene.4.Screening of transcription factors affecting the expression of FAM76B gene:The selected transcription factors that may affect the expression of FAM76B gene were screened in the cells established above,respectively.First,the expression vector carrying transcription factor was transfected into FAM76B-Luciferase-KI HEK293 cell line,and the empty vector was transfected into the same cell line as the control group.The results indicated that C-MYB and STAT5A inhibited the expression of FAM76B,whereas C-Ets-1 promoted the expression of FAM76B.Then,these three transcription factors were transfected into wild-type HEK293 cells,the results showed that C-MYB and STAT5A inhibited the expression of FAM76B,and C-Ets-1 promoted the expression of FAM76B,which was confirmed by the mRNA and protein expression level using Real-time PCR and Western blot respectively.The results above further demonstrated that luciferase expression level in the FAM76B-Luciferase-KI HEK293 cell line could reflect the expression level of FAM76B gene sensitively and accurately.5.Screening of small molecule chemical drugs affecting the expression of FAM76B gene:Our previous studies showed that knockout of FAM76B gene would lead to abnormal lipid metabolism in the liver of mice.Therefore,this new cell lines will be used to screen small molecule chemical drugs that can affect the expression of FAM76B.The results indicated that three drugs,Pioglitazone,Dihydroartemisinin and Tanshinone I,can significantly affect the expression of FAM76B.Pioglitazone could promote the expression of FAM76B,and Dihydroartemisinin and Tanshinone I could inhibit the expression of FAM76B.Then,the screened drugs were applied to wild-type HEK293 cells,respectively.The results showed that Pioglitazone could promote the expression of FAM76B,while Dihydroartemisinin and Tanshinone I could inhibit the expression of FAM76B indeed,which was confirmed by the mRNA and protein expression level using Real-time PCR and Western blot respectively.The results above indicated that the FAM76B-Luciferase-KI HEK293 cell line can be used as a reliable tool for screening small molecule drugs affecting the expression of FAM76B gene.In conclusion,we have obtained the following results in this study.1)Luciferase reporter gene was specifically inserted into the downstream of FAM76B gene using CRISPR/Cas9 genome editing technology,and the FAM76B-Luciferase-KI HEK293 cell line was successfully established;2)the cell line could be successfully used to screen transcription factors affecting the expression of FAM76B gene,which laid a solid foundation for the study on the function of FAM76B gene;3)the small molecule chemical drugs affecting the expression of FAM76B gene were successfully screened by this cell line,which provided a new idea and method for the functional study of FAM76B gene.The experimental results in this study showed that luciferase expression level could reflect the expression level of FAM76B gene sensitively and accurately in the FAM76B-Luciferase-KI HEK293 cell line.This new cell line can not only be used to study the regulation of FAM76B gene expression,but also be a useful tool for screening transcription factors or small-molecule drugs affecting the expression of FAM76B gene.Furthermore,the strategy by inserting reporter genes into the downstream of target genes with CRISPR/Cas9 genomic editing technology and using the expression level of reporter gene to reflect the expression level of target gene can also be widely used in the study of other target genes.