Construction of a LRP16 gene targeting vector and screening of homologously recombinant clone of embryonic stem cells / 中国组织工程研究

BACKGROUND: Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells.OBJECTIVE: To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP16 gene.DESIGN: Constructing an inserting inactivation target by inserting SA-RIES-β geo expression cassette.SETTING: Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA.MATERIALS: The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from lnvitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen Biotech Corp. pcDNA3.1(+) vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University.METHODS: The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction (PCR) screening. The SA-RIES-β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis.MAIN OUTCOME MEASURES: Clones with homologous recombination.RESULTS: The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES-β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted.CONCLUSION: A LRP16 gene targeting vector is constructed and a homologous recombinant is obtained.

The influence of anti-sense hTERT on the expression of VEGF and Flt in SGC7901 cells / 解放军医学杂志

Objective To investigate the effects of anti-sense hTERT on the expression of VEGF and the receptor in SGC7901 cells, and study the influence in angiogenesis and progression in gastric carcinoma. Methods SGC7901 cell line was transfected by the recombinant virus containing sense and anti-sense hTERT cDNA. Then the expression of VEGF-C and Flt-4 was observed with RT-PCR, distribution of cell cycles was determined with flow cytometry. The expression of VEGF-C and Flt-4 protein was assessed with immunohistochemistry. Result The distribution of cell cycle of antisense hTERT transfected SGC7901 cells was changed, and the mRNA and protein expression of VEGF-C and Flt-4 was significantly down-regulated. Conclusion Anti-sense hTERT can act as an agent for inhibiting VEGF-C and Flt-4 mRNA and protein level, and it blocks tumor angiogenesis and lymphogenous metastasis.

Cloning and Expression of Various Deletants of Gsalpha Gene in Escherichia Coli / 中国实验血液学杂志

Gsalpha gene mutation has been discovered in some human tumors. In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia. To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E. coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E. coli, respectively. As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies. The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro. The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.

Inhibitory effect of FHL2 on Id1-mediated suppression of proliferation and invasive capacity of breast cancer cells MCF-7 / 解放军医学杂志

Objective To investigate the inhibitory effect of four and a half LIM protein 2(FHL2) on inhibitor of differentiation 1(Id1)-mediated suppression of transcriptional regulation activity and Id1-promoted invasive growth of human breast cancer cells MCF-7.Methods The effect of FHL2 on Id1-mediated transcriptional repression in MCF-7 cells was determined by cotransfection and relative biluciferase assay.The cell proliferation was determined by MTT assay,and the invasive capacity of MCF-7 cells was determined by Transwell assay.Results The transcriptional repression effect of Id1 on basic helix-loop-helix(bHLH) factor E47-mediated transcription activity in MCF-7 cells,and Id1-promoted proliferation and invasive growth of MCF-7 cells were significantly suppressed by FHL2.Conclusion FHL2 can inhibit the proliferation and invasive growth of human breast cancer cells via repressing Id1-mediated functional activity.The results provide a basis for further investigating the functional roles of FHL2-Id1 signal pathway in the carcinogenesis and development of human breast cancer.