AIM To construct recombinase Cre expressing retrovirus expression vector and infect the mouse host cell in vitro . METHODS Cre gene was inserted into retrovirusexpression vector pMx IRES GFP and the packing cell line was transfected to produce retrovirus particles. Retrovirus particles infected NIH 3T3 cells in vitro , and the virus titer was determined by green lusaferase protein. RESULTS Recombinase Cre expressing retrovirus expression vector pMx IRES GFP Cre was constructed successfully, and the packing cell line was able to produce retrovirus particles. The virus titer was 10 5 cfu·mL -1 . CONCLUSION Recombinase Cre would be expressed by retrovirus expression system, and the retrovirus particles can infect the host cells effectively. This system will be helpful in studying the function of specific gene.
AIM:To clone and sequence the glucan bindin g protein B (GbpB) gene of S.mutans. METHODS:The bacteri al chromosomal DNA was isolate from S.mutans Ingbritt and the specific gene fragment was obtained by PCR with two gene specific primers.The segment was ins erted into pBluescript KS vector and the result plasmid was transformed into DH5 α.The positive clone was sequenced.RESULTS:The 1 300 bp spec ific fragment was obtained. The sequence of the gene encoding GbpB was consisten t with those of the references published.CONCLUSION:PCR techni que is effective method to clone the gene encoding GbpB from S.mutans.The re sult will help us to further studies about express and function of GbpB.
Objective To establish the transgenic mouse model of DSP.Methods Plasmid pcDNA3.1-CX was constructed by substituting promoter cβ-actin for CMV promoter of pcDNA3.1.And the ultimate transgenic vector,pcDNA3.1-CX-dsp,was constructed by cloning DSP coding sequence into pcDNA3.1-CX.The pcDNA3.1-CX-dsp plasmid was linearized and microinjected into the male pronucleus of the zygotes.The tail DNA of pups was tested by PCR and Southern blot.Results Seven hundred and seventeen(717) embryos were implanted to twenty-nine recipient pseudopregnant mice.Four of the sixty-seven pups carried the transgene.The establishment of the transgenic mouse line of DSP was under progress.Conclusion Founders of the DSP transgenic mouse were obtained successfully.
Haploinsufficiency of the runt-related transcription factor 2 (RUNX2) gene is known to cause cleidocranial dysplasia (CCD). Here, we investigated a complex, heterozygous RUNX2 gene mutation in a Chinese family with CCD and the pathogenesis associated with the variations. Genomic DNA extracted from peripheral venous blood was taken from the proband, her parents and 3 siblings, and 150 normal controls. Analysis of their respective RUNX2 gene sequences was performed by PCR amplification and Sanger sequencing. Pathogenesis associated with RUNX2 mutations was investigated by performing bioinformatics, real-time PCR, western blot analysis, and subcellular localization studies. We identified 2 complex heterozygous mutations involving a c.398–399 insACAGCAGCAGCAGCA insertion and a c.411–412 insG frameshift mutation in exon 3 of the RUNX2 gene. The frameshift mutation changed the structure of the RUNX2 protein while did not affect its expression at the mRNA level. Transfection of HEK293T cells with a plasmid expressing the RUNX2 variant decreased the molecular weight of the variant RUNX2 protein, compared with that of the wild-type protein. Subcellular localization assays showed both nuclear and cytoplasmic localization for the mutant protein, while the wild-type protein localized to the nucleus. Our findings demonstrated that the novel c.398–399insACAGCAGCAGCAGCA mutation occurred alongside the c.411–412insG frameshift mutation, which resulted in RUNX2 truncation. RUNX2 haploinsufficiency was associated with CCD pathogenesis. These results extend the known mutational spectrum of the RUNX2 gene and suggest a functional role of the novel mutation in CCD pathogenesis.
AIM:To express tuftelin and purify it from E.coli and prepare its antiserum. METHODS :The plasmid pPRoEX TM HTc and the sequence of Tuftelin cDNA(365 amino acid)cloning in the plasmid pBluescript KS+ were cut by the restricted endonucleas.Then the two fragment were ligased by T4 DNA ligasing and the expression plasmid of fusion protein was constructed.The product was transformed into E.coli . After the induce of IPTG, the E.coli was centrifuged and breakaged by SDS loading buffer. The purified tuftelin was mixed with incomplete adjuvant, and then used to immunize rabbits. RESULTS:The product of M r 42×10 3 fusion protein was in accordance with the anticipation. 8 weeks later. the antiserum titer of 1:100 000 could be showed by the test of Western blot. CONCLUSION :The tuftelin is expressed successfully and the antiserum is prepared.
AIM: To observe the expression of cbfa1 in mouse tooth germs, to clone and sequence the cDNA of cbfa1 from mouse tooth germs. METHODS: Total RNA was isolated from newborn mouse tooth germs tissue and reverse-transcribed into cDNA. The desired DNA product was obtained by PCR with two cbfa1 specific primers. The segment was inserted into pGEM-5zf vector and the positive clone was sequenced by Takara Corporation. RESULTS: cDNA of cbfa1 was obtained from mouse tooth germs tissue. The sequence was consistent with that displayed on Pubmed. No mutation was found. CONCLUSION: This study provides the evidence of expression of cbfa1 in mouse tooth germs tissue.
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