Construction Of Targeting Vector For Conditional Knockout Of NPM & Selection And Identification Of Targeted ES Cells

Conditional gene knockout is developed as being a new strategy with combination of site-specific recombination mediated by recombinase on the basis of tranditional gene targeting, in which modification on specific gene was confined in certain kind of cell, tissue even in special development period of mice. In conditional gene targeting system with Cre/LoxP recombination, the important functional domains of targeting gene were floxed by two LoxP sites, usually being inserted into some introns. And therefore Conditional knockout mice show normal phenotype. After mating with mice with special Cre expressoin in some cells or tissue, target gene will be deleted by Cre/LoxP recombination in the specific region. The gene will be kept intact expression in other tissues. So it is one good tool to study the function of some genes with deletion-lethality during embryo development.NPM (nucleophosmin),one of the multifunctional phosphoproteins, mainly locates in nucleoli and shuttles rapidly between the nucleus and cytoplasm. It can involve in transporting ribosomal precursor and centrosome duplication to regulate cell cycle process and organism development. In human NPM was mapped on 5q35. It contains 12 exons with the size of about 23 kb. From the N terminus, this protein contains one hydrophobic segment that is involved in oligomerization as well as chaperone activity, followed by two acidic stretches that are important for binding to histones, the remains and C-terminal domain which contains basic regions involved in nucleic-acid binding. NPM also contains nuclear localization signal, a nuclear export signal and nucleolar localization signal and multiphosphorylation sites.NPM was implicated in human tumorigenesis. NPM was frequently overexpressed in solid tumours of a diverse histological origin, such as gastric, colon, ovarian ,prostate and liver carcinomas, and genetic alterations occured frequently in haematopoietic tumours. It seems to function as either an oncogene or a tumour suppressor. Further investigation can address that what role of NPM is playing during tumor genesis and development. Target disruption of NPM gene resulted in embryonic lethality at mid-gestation. To study the function of NPM in vertebrate organgenesis and tumorigenesis, The model of NPM conditional gene knockout mice is very important. The first step was to make the conditional gene targeting vector. In the constructor, 2-6 exons of NPM were floxed by two LoxP sites.A 13kb genomic DNA fragment containing NPM full length gene, obtained from BAC by subcloning, was inserted into pBS KS [pBluescript II KS(+)] vector and named pBS KS-NPM. A 1.8kb DNA fragment containing exon 1, obtained from pBS KS-NPM by subcloning, was inserted into pBS KS vector which was used as the 5'homologous arm. A 8kb DNA fragment containing exon 2-9, obtained from pBS KS-NPM by subcloning as well, was inserted into pBS KS vector which was used as the 3'homologous arm. Then 3'homologous arm was inserted into pGEX-KG vector and named pGEX-3'arm to obtain suitable enzyme sites, and the LoxP3 was inserted into pGEX-3'arm at Kpn I restriction enzyme site within intron 6, this fragment was used as the final 3'homologous arm. Afterward 5'homologous arm and 3'homologous arm were cloned into classical targeting vector pLoxP I, respectively. Finally NPM conditional gene targeting vector were obtained, in which exons 2-6 were anchored by LoxP. The targeting vector was confirmed by PCR, restriction enzyme digestions and partial sequencing analysis, named pLoxP-NPM.Linearized by Not I targeting construction was introuduced into ES cells by electroporation. 53 ES clones were picked up after cultured in G418 and Gancyclovir for 7 days. The clonies were amplified and subjected to genomic DNA preparation. And 21 clonies were confirmed by PCR identification. The genomic DNA were digested with Hind III and used for Southern blot analysis. A probe outside the 3'homologous arm was used for hybridization.13 kb positive band was found in wild type.The band was shift down from 13kb to 7232bp in NPM conditional targeted allete because a new Hind III site was introduced into the intron along with the LoxP site. Targeted ES cells are the basis to generate NPM conditional gene knockout mice.