Construction Of Recombinant Telomerase Reverse Transcriptase Gene Expression Vector And The New Methods To Detect Telomere Length

Partâ… IntroductionHuman telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase. It is also responsible for addition of TTAGGG repeats to the telomere ends of the chromosome, which is important in maintaining cell immortality.In order to investigate the main functions of hTERT gene which promoted cell proliferation and telomerase activity, we applied gene transfer technique to promote the over-expression of extrinsic hTERT gene in human umbilical vein endothelial cells (hUVECs) and observed the effects on cell proliferation. It also preliminary confirms that telomerase activity and cell proliferation are closely related and settles a basis for establishing immortalized fibroblast of cells. Partâ…¡Overexpression of hTERT Promotes the Proliferation of Human Umbilical Vein Endothelial CellsObjective: In order to investigate the main functions of hTERT gene which promoted cell proliferation and telomerase activity, we applied gene transfer technique to promote the over-expression of extrinsic hTERT gene in human umbilical vein endothelial cells (hUVECs) and observed the effects on cell proliferation.Methods: The eukaryotic expression vector pEGFP-C1-hTERT was constructed with PCI-neo-hTERT and pEGFP-C1 plasmids,and the accuracy of human telomerase reverse transcriptase(hTERT)gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.Then the plasmid was transfected into hUVECs by liposome-mediated transfection .Results: The results indicated that fluorescence microscopy image had been taken after transfection.The cells group which transfected pEGFP-C1-hTERT (hUVEC- hTERT) were growing faster than the group which transfected pEGFP-C1 (hUVEC-Null) and control (hUVEC) after 72h. RT-PCR, Immunocyto chemical assay (ICA) and TRAP-PCR-ELISA showed that hTERT mRNA and protein expression were remarkably increased in transfected hUVECs.Conclusion:In conclusion, this study successfully constructed recombinant plasmid pEGFP-C1-hTERT and extrinsic hTERT gene transfection to hUVECs can be expressed; telomerase can be reconstructed and promoted the proliferation of cells.It also preliminary confirms that telomerase activity and cell proliferation are closely related and settles a basis for establishing immortalized fibroblast of cells. Key words: hTERT,eukaryotic expression vector,hUVECs,transfection Partâ…¢Construction of Recombinant Telomerase Reverse Transcriptase Gene Lentiviral Expression Vector and Virus PackagingObjective: The aim of this study is to construct lentivirus vector containing GFP reporter gene drived by human telomerase reverse transcriptase (hTERT) gene and to research a high-titer lentiviral packaging system, then observed the expression of hTERT gene.Methods: Digested the plasmid PCI-neo-hTERT with double enzyme digestion and subclonde hTERT into the lentiviral vector pCDH-copGFP, to generate the lentiviral expression vector pCDH-hTERT. The accuracy of hTERT fragment was confirmed by double enzyme digestion and DNA sequencing analysis. Recombinant lentiviruses were produced by 293T cells following the co-transfection of pCDH-hTERT, with the packaging plasmids pCDH-PACK-GAG, pCDH-PACK-REV and VSV-G which are the 3rd generation of lentiviral vector systems containing green fluorescent protein gene (GFP). Virus supernate was collected and concentrated,then tested the titer of virus.The resulting recombinant lentiviruses which carrying hTERT were used to infect target cell lines.GFP ,hTERT mRNA and telomerase expression in 293T and hUVEC were dectected by fluorescent microscope , RT-PCR,Western blotting and TRAP-PCR-ELISA.Result:Plasmid pCDH-hTERT carried the correct hTERT gene.The recombinant lentiviruses pCDH-hTERT which carried hTERT could be produced by co-transfection of pCDH-hTERT and packaging plasmids to 293T; The recombinant lentiviruses which carried hTERT could transfect and deliver hTERT gene to 293T and hUVEC, hTERT mRNA and protein expression were remarkably increased in transfected cells.Conclusion: This study successfully constructed recombinant plasmid pCDH-hTERT, the recombinant lentiviruses can deliver target gene hTERT and have high infection efficiency. Extrinsic hTERT gene can reconstruct telomerase and settle a basis for establishing immortalized fibroblast of cells. Partâ…£The new, rapid and sensitive methods using nanogold probes technology preliminary to detect telomere lengthObjective: Southern hybrid technology applied by comparative study of telomere length of particularly long-lived species in Guangxi, explore whether species exist similar longevity mechanism and evaluate the test whether suitable for other long-lived species.The study tries to illustrate the feasibility of the new method.DNA hybridization resonance scattering detection using nanogold probes as nanocatalyst, in order to explorate preliminary detection telomere length.Methods: Choose the common longevity species tortoises, cobra grass in guangxi as the research object, we applied southern hybrid technology to test different age of longevity species telomere length and compare telomere length. Nanogold particles in size of 10nm were used to label the thiol-capped single strand DNA aptamer (SH-ssDNA) to obtain an aptananogold probe (AussDNA) 5'-(CCCTAA)5(CH2)3SH-3'for target DNA (tDNA). To optimize the nanogold particles and an aptananogold probe connection and the oligonucleotide microarray telomere hybrid reaction system conditions, and explore whether the new method using nanogold probes to detect telomere length is feasible.Result: Southern hybrid technology applied by testing telomere length exist false positives and false negatives, each group were not statistically significant comparison. And then we successfully complete the nanogold particles and an aptananogold probe connection,But it is still not find suitable hybrid reaction conditions, have not been able to establish the new method using nanogold probes to detect telomere length. Conclusion: Southern hybrid technology for people using specific probe, is not suitable for other long-lived species, so the new, rapid and sensitive method must be established to detection telomere length according to different species. We also find that the oligonucleotide microarray telomere hybrid reaction system conditions is a difficult problem, how to solve with sample telomere biological probe hybrid reaction system will be the subsequent experiment primary problem solving.