Construction And Identification Of Recombinant Adenovirus Vector Of Zinc Finger Protein ZBTB20

Regulation of gene transcription is essential for gene expression and organism’sdevelopment. Zinc finger protein belongs to a class of the DNA-binding protein withfinger-like domain, and plays a positive or negative role in gene expression regulation,cell differentiation, embryonic development, tissue function and body metabolism. Thezinc finger protein ZBTB20, originally identified from human dendritic cell, is firstfound and reported by Zhang Weiping group. ZBTB20is a741-amnio-acid protein andcontains unique N-terminal BTB/POZ domain (interact with DNA) and5C2H2C-terminal DNA-binding zinc finger domains. ZBTB20global knockout mice showobvious obstacle in postnatal growth retardation, metabolic dysfunction and memorydisorder,, indicating that ZBTB20plays a nonredundant role in the control of postnataldevelopment and metabolism. However, its biological function, the downstream targetgenes and the signal pathways mediated are still far from clear.The recombinant adenovirus is a kind of integrated double-stranded DNA virus,which has no carcinogenicity because there is no mutation segment inserted. It has beenparticularly appealing in gene transfer due to its ability to infect both the dividing andnon-dividing cell. Meanwhile, it is easy to construct, concentrate, purify and finallyachieve high-titer adenovirus. Our earlier studies have shown that ZBTB20plays animportant role in liver lipid metabolism and the development of hepatic carcinoma, sothe recombinant adenovirus vector with liver targeting is undoubtedly the best tool forfurther study of ZBTB20biological function and its mechanism in the liver. For this purpose, we constructed two kinds of recombinant adenovirus-adenovirus bearingZBTB20or ZBTB20short hairpin RNA (shRNA) to study the physiological andpathological functions of ZBTB20by overexpression or interference strategy.We cloned the ZBTB20cDNA fragment with FLAG tag or shRNA targetingZBTB20into pAdTrack-CMV vector and introduced the linearized vector into AdEasierbacterial cells for generating of homologous recombinant adenovirus plasmids, andtransfected such plasmids into HEK-293packaging cells for the production of therecombinant adenovirus. By this method, we got two recombinant adenoviruses,AdZBTB20and AdZBTB20-shRNA. After three rounds of amplification in HEK-293cells, the recombinant viruses with high-titer were concentrated and purified with CsClgradient centrifugation. The viral titer was then determined by measuring the opticaldensity at260nm and TCID50. And the gene transfer ability of AdZBTB20orAdZBTB20shRNAwas determined in vitro and in vivo.Using the primary hepatocytes, the expression and activity of ZBTB20protein wasdetermined by Western blot and reporter gene assay after Ad-ZBTB20infection.We found that the mRNA and protein levels of ZBTB20were dramatically upregulated,with the activity of inhibiting the transcription of its target gene alpha-fetoprotein. Andthe overexpression of ZBTB20was also determined in the liver tissue fromliver-specific ZBTB20knock-out mice injected with Ad-ZBTB20via tail vein.Meanwhile, the expression of endogenous ZBTB20protein in primary hepatocytes andliver tissue from wild type mice was significantly decreased by AdZBTB20shRNAinfection.In conclusion, we successfully prepared the recombinant adenovirus Ad-ZBTB20which could overexpress ZBTB20protein with transcription regulation activity, and therecombinant adenovirus AdZBTB20shRNA which could downregulate endogenousZBTB20level in vitro and in vivo. The two adenoviruses will provide a useful tool forfurther investigation of the biological role of ZBTB20.