| Zinc finger proteins are a class of proteins containing zinc finger structures,in which surrounding amino acid residues are non-covalently linked to each other by zinc ions to form a stable DNA binding zinc finger domain.Decades after the discovery of zinc finger structures,more than 10 types of such structures have been discovered.Among them,the total number of proteins containing the initially discovered TFIIIA type zinc finger domain is about 1% of the human gene product.Based on the structure and function of zinc finger proteins,they can be divided into 9 categories: Cys2His2,Cys8,Cys6,Cys3 His Cys4,Cys2 His Cys,Cys2 His Cys5,Cys4,Cys3 His and Cys4 His Cys3.According to the spatial structure of residues around the zinc ion,zinc finger proteins can also be divided into C2H2-like zinc fingers,Treble clef zinc fingers,Zinc ribbon zinc fingers,Gag knuckle zinc fingers,Zn2/Cys6 fingers,TAZ2 domain like zinc finger,Zinc binding loops zinc finger,and Metallothionein zinc finger,in which the first three groups contain most of the zinc fingers.Since zinc finger structures were first discovered in Xenopus oocytes,thousands of zinc finger proteins have been identified.The diversity of structures gives them powerful functions,based on their function of transcription and post-transcriptional regulation,protein folding and assemble,and DNA and RNA recognition,they are involved in regulating embryonic development,natural immunity,and tumor formation and development.Zinc finger protein,as the most widely distributed transcription factor,plays an important role in regulating the function of organisms.ZKSCAN3 belongs to the C2H2 type zinc finger protein and was reported earlier in gene screening studies related to cell proliferation.Subsequent studies found that ZKSCAN3 was highly expressed in colorectal cancer tissues while low expressed in adjacent tissues,and the results of immunohistochemistry staining also showed that ZKSCAN3 expression level was higher in invasive tumor tissues,when compared with non-invasive tumor tissues.In addition,knockout of ZKSCAN3 significantly inhibited the growth of colon cancer cells,overexpression of ZKSCAN3 promoted cell growth and increased drug resistance.Further studies identified KRDGGG as the DNA binding motif of ZKSCAN3,upregulating the expression of multiple genes associated with cell proliferation,cell migration,angiogenesis,and proteolysis,and promote tumor development in other tumor types such as bladder cancer and multiple myeloma.In addition,ZKSCAN3 is considered to be an important transcriptional repressor of autophagy and inhibits a series of genes related to autophagy and lysosome production.ZKSCAN3 belongs to a family of transcriptional regulators and its structure consists of a zinc finger domain,a KRAB(Krüppel-associated box)domain and a SCAN domain.The KRAB domain is a potent transcriptional repression unit that is generally thought to bind to co-repressors and/or transcription factors through protein-protein interactions.In vivo and in vitro experimental results suggest that overexpression of ZKSCAN3 can promote tumor cell growth.In contrast,knockout of ZKSCAN3 resulted in cell growth arrest and accelerated aging.In recent years,the maturity and development of CRISPR technology have made it an important technique for gene editing and have shown great potential in gene therapy.Using this technology to knock-out key molecules in tumor cells can inhibit the growth of tumors and their malignancy,and have also achieved good outcomes in the treatment of tumors.However,the existing delivery methods such as lentivirus transfection,adeno-associated virus transfection,or electroporation,lipidbased transfection,etc.,cannot completely solve the problem of targeted delivery,and there are still problems in the CRISPR system delivery to the organism.Therefore,despite considering the benefit of ZKSCAN3 knock-out in tumor tissues,we still need to examine the effect of knock-out in normal tissues on organisms.In addition,CRISPR technology has a lower knock-out efficiency with the length of the knockout sequence increases,how to select the appropriate targeting sequence based on the characteristics of ZKSCAN3 still need to be answered.OBJECTIVE: To construct Zkscan3 knockout mice,detect the expression of Zkscan3 in vivo by reporter gene expression,and detect the physiological and pathological changes of Zkscan3 knockout mice to determine the effect of Zkscan3 knockout in normal tissues and organs on mice.In addition,according to the structural characteristics of Zkscan3,the function of recombinant proteins lack different domains was studied,and clarify the optimal targeting interval of this molecule.METHODS: Insert EGFP reporter gene expression box in place of the Zkscan3 start codon to replace its expression.At the same time,insert the loxp site at both ends of the EGFP expression frame.Through mating with the corresponding Cre tool mouse,we can recover Zkscan3 expression in specific tissues or organs.The expression of EGFP in tissues and organs was detected by flow cytometry and tissue immunofluorescence to indirectly understand the expression range of Zkscan3 gene in tissues and organs.In addition,the mouse Zkscan3 gene was cloned,and lentivirus vector was constructed and transfected into CT26 cells.Zkscan3 truncations with domains deleted were fused with GFP,forming fusion protein,flow cytometry and cellular immunofluorescence techniques were used to investigate the localization and expression of fusion proteins with different domains.Finally,through RNA-SEQ technology,we have a deep understanding of the features and functions of Zkscan3 that lack functional domains,as well as its influence and regulation on other genes in the cell.In addition,the potential targets of Zkscan3 were searched to compare their similarities and differences in function in humans and mice.RESULTS: Zkscan3 knockout mice were successfully constructed and the expression of GFP in each mouse tissue was initially observed.Compared with human ZKSCAN3 which is highly expressed in the bone marrow,lymph nodes,tonsils,lung,gallbladder,stomach,small intestine,colon,bladder,epididymis and placental tissue,mouse Zkscan3 is expressed in organs like skin,heart,lung,liver,spleen and kidney,etc.Loss of Zkscan3 in each organ did not have a lethal effect,and the survival of the knockout mice was not different from that of the wild type mice.Therefore,non-specific knockout of ZKSCAN3 in normal tissue organs is safe.In addition,according to the expression of the fusion protein formed by Zkscan3 truncations lacking different domain and GFP,fusion protein with zinc finger domain can be located in the nucleus,while the SCAN domain and/or KRAB domain is located in the cytoplasm.This shows that the zinc finger structure region is the best target range for ZKSCAN3.In particular,cells expressing fusion proteins with zinc finger domain showed significantly lower expression rates than cells expressing corresponding fusion proteins with zinc finger domain.In addition,the RNA-SEQ analysis of the zinc finger domain of the zinc finger indicates that the zinc finger domain itself can upregulate the expression of VEGF,and regulate the expression of genes associated with other target genes,indicating that the domain has the ability to act independently.Combined with RNA-SEQ analysis of full-length Zkscan3 expressing CT26 cells,we screened and verified 18 potential target genes.Real time-PCR results showed that Ccl2 may represent another downstream target gene of Zkscan3. |
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