Knock-out Mice Model Establishment And Function Analysis Of Zkscan3

ZKSCAN3 is a zinc finger protein with KRAB domain and SCAN domain, which locus on chromosome 6p22.1 region in human. ZKSCAN3 contains KRAB, SCAN and C2H2-type zinc finger domain. Among them, the KRAB domain functions as a transcriptional repressor when binds to template DNA, while the SCAN domain may play an important role in assembly and function of C2H2 zinc finger proteins, C2H2 zinc finger domain binds to DNA, RNA, protein and other small molecules, and different zinc finger sequences and links determine the binding specificity of zinc finger proteins. It is showed that ZKSCNA3 binds to KRDGGG motif and up-regulate genes associated with cell growth, migration, angiogenesis and proteolysis, such as MST1 R, MEK2, Ras GRP2, IGF-2, integrin β4, VEGF, MMP26 and PRSS3. Since these gene products play an important role in the tumor growth progress, ZKSCAN3 may represent a potential target for cancer treatment. Data also shows that, ZKSCAN3 significantly promote the development and metastasis progress of colorectal carcinoma, multiple myeloma and prostate carcinoma. Given its important role in tumor pathology, it is of great significance to study the function and potential mechanism of ZKSCAN3 through zkscan3 knock-out mice model. In this project, we established a zkscan3 knock-out mouse model to systematically study the role of zkscan3 in mouse development and its physiological functions. Since the current KO model expresses truncated zkscan3 m RNA, we may expect the KO model still has partial zkscan3 functions. Accordingly, we have initiated generating a novel zkscan3 KO model based on a different strategy to destroy zkscan3 gene completely, which may provide us a better model to study zkscan3 gene function thoroughly.Methods:1. In order to investigate the function of zkscan3 in development, zkscan3 knockout mice was achieved by crossing zkscan3 floxed mice with Ube-Cre mice, thus we get zkscan-/- mice in which the exon2 of zkscan3 is deleted.2. PCR, RT-PCR were used to identify zkscan3 deletion3. Statistic the genotype ratio and sex ratio of zkscan3-/- mice after birth4. Statistic the growth curve of zkscan3-/- mice from D10 to D40 after birth.5. H&E staining was used to analyze different organ development of zkscan3-/-mice6. Blood analyzer was used to test the hematopoietic function after zkscan3 deletion7. Di I-labeled RBC was injected i.v into zkscan3-/- mice to monitor the metabolism of RBC in vivo and to check the function of macrophage in mice spleen.Results:1. PCR showed that the exon2 of zkscan3 gene is deleted.2. RT-PCR indicate that there’s m RNA product in zkscan3-/- mice while lack the part of exon 23. The ratio of each genotype fit well with Mendel’s law, indicate that zkscan3 deletion has few impact on mice embryo development.4. There’s no significant difference between the mice body weight of each genotype from D10 to D40 at postnatal stage.5. H&E staining showed no significant difference between organs of each genotype, however, hemosiderin-like deposition appeared in part of zkscan3-/- mice spleen.6. Blood analysis result showed no significant statistical difference between the blood analysis result of each genotype7. Di I-labeled RBC concentrated in the red pulp area of mice spleen, and merged with hemosiderin-like deposition, thus indicate that the hemosiderin-like deposition is caused by RBC metabolism desfunction.Conclusion:We have generated zkscan3-/- mice successfully, in which the exon2 of KO mice genome is deleted. However, zkscan3 transcript which lack exon2 still appears, whether or not there’s corresponding protein product is unknown. The genotype ratio of zkscan3-/- mice is normal, indicating that zkscan3 knockout has no lethal impact on mice embryo development. There’s also no significant difference between the growth curve of zkscan3 KO mice and WT mice, suggesting that zkscan3 knockout mice have no effect on their growth. In addition, H&E staining is applied to investigate the organ development in zkscan3-/- mice, but the majority of the organs are normal, only there’s hemosiderin-like deposition in the dorsal area of zkscan3-/- mice spleens. The function analysis of spleen suggests that zkscan3 may be associated with phagocytosis of mice macrophage.