Identification And Phenotypic Analysis Of Ndrg2-conditional Knockout And Ndrg2shRNA Transgenic Mice

The human NDRG2（N-myc downstream regulated gene2）was first cloned from thenormal human brain cDNA library in our lab in1999. NDRG2is located on14q11.2,including16exons and15introns.. Its cDNA is2024bp in length that encodes a40kDaprotein with357amino acid residues. The NDRG gene family consists of4members：NDRG1、NDRG2、NDRG3and NDRG4. Although highly conserved in amino acid identityand structure，they have different patterns of tissue distribution, which suggests that theymay exert similar function in different diverse tissues.NDRG2plays an important role in the tumorigenesis, embryo development, celldifferentiation,stress responses, and nervous system degenerative diease such asAlzheimer’s disease. Previous study showed that NDRG2could be regulated by varioustranscription factors such as Myc﹑p53, Hif-1﹑ER. It was also reported that NDRG2interects with MSP58﹑PRA1and involves in the activation of MAPK and β-cateninsignal transduction pathway. But these research in vivo are rare almost of them are limtedon cellular and molecular level.During the last decade, Knockout and transgenic animals are likely to become basicstrategy for analyzing gene function at whole animal level in vivo. The technology of geneknockout is based on the homologous recombination. Exogenous gene is transfered intoembryonic stem cells to replace target gene on the genome for completely deletion byhomologous recombination. Conditional gene knockout is a relatively new technique, an offshoot of gene knockout technology. The most commonly used technique for conditionalgene knockout is the Cre-Lox recombination system. Spatial and temporal elimination ofspecific target gene rely on Cre’s ability to recognize and cut a pre-designated DNAsequence that has been flanked by directly repeated copies of the loxP recombination site.Gene knockdown refers to an experimental technique by which expression of target genesare reduced. The reduction can occur either through genetic modification or by treatmentwith a reagent such as a short DNA or RNA oligonucleotide that has a sequencecomplementary to either gene or an mRNA transcript. This method is simple and easy tooperate. It can make the specific target gene reduceing at different lever in vivo. To furtherexplore the function of NDRG2in vivo，Ndrg2-conditional knockout and Ndrg2shRNAtransgenic mice was generated. The identification and basic phenotype analyses wereperformed on Ndrg2mouse models.In the first part, to further study the function of NDRG2in vivo Shanghai BiomodelOrganism Science&Technology Development Co.,Ltd was authorized to generateNdrg2-conditional knockout. Genotype of the mouse was dentified byPCR. The knockouteffiency of Ndrg2was analized in vivo by Realtime PCR、 Western blot、 andimmunohistochemistry. Basic phenotype analyses were conducted in the Ndrg2knockoutmice. The expression of other Ndrg family members in knockout mic was detected withRealtime PCR. Results：the gene knockout carrier is successful inserted in mice genome.Ndrg2is completed knockout in various tissue of knock out mice at mRNA and proteinlevel. The tissue structure of muscle,kidney,pancreas,spleen,lung are normal.Meanwhile,the expression of Ndrg1、 Ndrg3and Ndrg4were enhanced various tissue ofNdrg2-conditional knockout in vivo.In the second part, to explore the role of NDRG2in vesselendothelium,Ndrg2-knockout mice liver organ was observed. The vessel endothelium ofwas observed to be thickened in liver tissue, compared to control group. The expressionendothelial cell’s marker molecules,,such as VWF、CD31and CD34increased dramaticlly.NDRG2overexpress and knockdown HUVEC cell line were generated by lentivirusinfection. The expression of NDRG2in HUVEC was analized by Realtime PCR andWestern blot. The effect of NDRG2on cell proliferation was detected by FACS inHUVEC. Results：We successfully build HUVEC which stablly overexpress and interfere NDRG2.Compared with control group, the expression of NDRG2increased24times, inNDRG2overexpression cells. In the group of NDRG2interference, the expression ofNDRG2was reduced50%.The cell proliferation rate was found to decressed dramaticllyin the NDRG2overexpression cells, compared with control group. Whereas cellproliferation rate of NDRG2interference cells were incressed dramaticlly.In the third part, Ndrg2shRNA transgenic mice was generated based on RNAitechnology, genetype of mice was analized by PCR. The effect of Ndrg2knockdown wasdetected by Realtime PCR,Western blot. Results: Ndrg2specific interference carrier isintegrated into the genome of Ndrg2shRNA transgenic mice, and could be detected attranscription level, but the expression of Ndrg2was not reduced obviously in Ndrg2shRNA transgenic mice, compared with wild type mice.Conclusion: Ndrg2gene has been completely knockout in Ndrg2-conditionalknockout mice. Inactivation of Ndrg2gene does not lead to an observed abnormalphenotype with naked eyes. While the vascular endothelial becomes thickened in the livertissue of Ndrg2-conditional knockout mice. Further investigation showed that theoverexpression of NDRG2result in decreased proliferation of HUVEC cells. Moreover,the expression of Ndrg1, Ndrg3and Ndrg4increased significantly in Ndrg2-conditionalknockout mice. Ndrg2specific shRNA has been integrated into the host genome and couldbe detected at transcription level. However, the Ndrg2has not been knockdown efficiently.Taken together,Ndrg2shRNA transgenic mice was not generated successfully.