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Construction of Targeting Vector for Conditional Knockout of NPM & Selection and Identification of Targeted ES Cells

Author: ZhangHongSheng
Tutor: GuanXiong；HuBaoCheng
School: Fujian Agriculture and Forestry University
Course: Biochemistry and Molecular Biology
Keywords: NPM Conditional gene targeting Cre / LoxP
CLC: Q782
Type: Master's thesis
Year: 2009
Downloads: 100
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Abstract

Conditional gene targeting is on the basis of the conventional gene targeting, combined recombinase-mediated site-specific recombination technology developed from a gene targeting technology. It can be the modification of a specific gene will be limited to specific types of cells, tissues and even the specific stages of mouse development. Cre / LoxP conditional gene targeting system, usually by means of homologous recombination two LoxP sequence in the intron of the target genes important functional domains on both sides, want to get normal phenotype conditional gene targeting mice; then this expected to be tissue-specific knockout mice, mice with cells or tissue-specific expression of Cre recombinase transgenic mice were crossed. Wherein the Cre recombinase is only expressed in a particular cell or tissue, and the Cre / LoxP sites dependent recombinant knockout of the cells of the target, while the other cell types in the absence of the expression of Cre recombinase, the target genes are not will be removed while still maintaining the original expression patterns. Thus, as we study certain deletions result in embryonic lethality gene function provides a good research tool. NPM is a multifunctional protein, mainly located in the nucleolus area, shuttling between the nucleolus, nucleoplasm and cytoplasm, involved in the transport of precursor synthesis and centrosome replication process of the ribosome, thereby regulating cell cycle progression with the development of living organisms. Human NPM gene is located on chromosome 5q35, total length of about 23Kb, and contains 12 exons. Entire protein comprises three functional domains: oligomerization chaperone functional domain, histone binding domain and a nucleic acid binding domain, the positioning signals and having a series of sequences (such as the nuclear export signal, nuclear localization signal, and nucleolar localization signal ) and phosphorylation sites. The results show that in the blood tumor NPM frequently mutated, suggesting that it might be a tumor suppressor gene. The study found that overexpression in some tumor cells (such as stomach cancer, colon cancer, ovarian cancer, prostate cancer, and liver cancer), and may also hinted NPM is an oncogene. What NPM how to play in the development of these tumors occur remains to be further research. View of the NPM completely gene knockout mice embryos in the Developing death. In order to further study the NPM adult biological function of various tissues and organs and tumor development, we are trying to establish the primary task is to build the the NPM conditions knockout vector NPM conditional knockout mouse model established mouse model, based on NPM N end and the importance of the intermediate region as well as the feasibility of the experimental operation, the two LoxP sequences were placed on both sides of the functional domains of the N-terminal and the middle of the NPM gene sequence intron. First, we cloned from the mouse genome BAC plasmid section contains the NPM entire genome sequence, including 13Kb fragment, connected to the pBS KS [pBluescript II KS ()] vector, named for the pBSKS-of NPM. We then again from the segment sequence cloned the size of the two fragments of 8 kb and 1.8 kb, respectively, as the NPM conditional gene targeting vector the 5 'and 3' homology arms, are respectively connected to the pBS KS vector, select Insert Cloning of the correct direction. 3 'homologous arm after then cloned into the vector pGEX-KG, carrier Kpn I single restriction sites inserted at the intron 6 LoxP3. Last two homology arms were cloned Universal the targeting vector pLoxP I on, forming a 2-6 NPM gene exon the LoxP sequences anchored conditional gene targeting vector, named to as pLoxP-of NPM. PCR screening, restriction enzyme digestion and sequencing results prove NPM conditional gene targeting vector is consistent with the expected structure. Finally, the targeting vector Not I linearized shock transfected ES cells, and resistant clones were picked 53 filters after 7 days in a selective medium containing G418 and Gancyclovir. By PCR preliminary identification of 21 positive clones, need further of these positive clones were amplified after extraction of genomic DNA was digested with Hind III, with the targeting vector containing the 3 'end of the outer probe to Southern blot analysis, and the wild-type will appear 13kb strip, while NPM NPM intron LoxP sequence due to the insertion to carry a Hind III site, the target allele in the target cells will appear band of 13kb and 7232bp two of. ES cells and target successfully laid the foundation will further establish the NPM tissue-specific knockout mouse model.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering) > Gene vector
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