Preliminary Study On The Function Of The Novel Gene TM4

PartⅠThe construction of TM4 gene recombinant eukaryotic expression vector and the effect of TM4 on HEK293 cell proliferationObjective:To construct recombinant eukaryotic expression vector with TM4 gene and over-express TM4 gene in HEK293 cells in order to observe the effect of TM4 on cell proliferation.Methods:The full-length cDNA of TM4 gene was amplified from pDrive-TM4 recombinant plasmid and was further subcloned into eukaryotic expression vector PDC315 and PDC315-GFP.TM4 gene in PDC315-TM4 and PDC315-GFP-TM4 recombinant plasmids were confirmed by sequencing and further cell transfection.Results:PDC315-TM4 and PDC315-GFP-TM4 recombinant eukaryotic expression vectors were successfully constructed and had been confirmed by sequencing and further cell transfection.Proliferation was increased about 43%at 72h post transfection of PDC315-TM4 in HEK293 cell.Conclusions:TM4 gene recombinant eukaryotic expression vector was successfully constructed.TM4 stimulates the proliferation of HEK293.PartⅡPreparation,Breeding and Identification of mouse model of TM4 gene mutation Objective:To prepare the mouse model of TM4 gene mutation by gene trap.Methods:The mouse model of TM4 gene mutation was produced using gene-trapping approach.The mutation of the gene was finally confirmed at the mRNA and protein level by semi-quantitative real-time PCR and Western-Blotting analysis of TM4 expression.In addition,the mutation was crossed the 4th generations into the C57BL/6 genetic background by mating between male heterozygous mice with black fur and female C57BL/6J mouse,which called F2,F3 and F4,respectively.Results:Homozygous mice were obtained successfully.The gene-trapping vector has been successfully inserted into intron of TM4 gene,which leads to mutation in F2,F3 and F4 mouse.The levels of mRNA and protein of TM4 gene in the homozygous mutant mice were significantly lower than those in wild-type mice.Conclusions:The mouse model of TM4 gene mutation was obtained successfully.PartⅢThe investigation of phenotypes in the TM4 gene mutant mouseObjective:To observe the phenotypes in the TM4 homozygous mutant mouse.Methods:The fasting blood glucose,random blood glucose,the blood glucose in starvation condition were measured and blood pressure of the homozygous mutant mice and wild-type mice were examined separately.Mouse abdominal aortas were isolated under the microscope and the Phenylephrine and acetylcholine-stimulated reactive of abdominal aortic rings were tested.Results:(1) The fasting blood glucose,random blood glucose,and blood glucose in starvation condition were 5.54±1.49mmol/L,7.56±1.08mmol/L and 5.19±1.13 mmol/L in group of TM4 gene homozygous mutant mice(n=8) respectively,and 5.29±1.14 mmol/L,6.88±0.75 mmol/L,5.04±0.72 mmol/L in wild-type group(n=8) respectively.The levels of blood glucose in group of TM4 homozygous mutant were little higher than those in wild-type group(P＞0.05).(2) Blood pressure:The systolic blood pressure of TM4 gene homozygous mutant mice(n=5) was 127.14±4.45mmHg and the diastolic blood pressure was 98.5±10.21mmHg.The systolic blood pressure was 144.8±5.35mmHg and diastolic blood pressure was 108.4±11.28mmHg in wild-type mice(n=4).The systolic blood pressure of homozygous mutant group was significantly lower than those in the wild group(P = 0.037).(3) There was no significant vascular tension difference between TM4 homozygous mutant mice(n=3) and wild-type mice(n=5) after KPSS stimulation.The vascular tension was increased 9.71±1.26 mN and 8.82±1.78mN in TM4 homozygous mutant mice(n=3) and in wild-type mice(n=5) when treated with Phenylephrine (P＞0.05).Considering the maximum phenylephrine contraction as 100%,the percentage change of vasodilatation was calculated after treating with Ach and was 17.37%±7.38%and 36.70%±20.5%in the two groups respectively.The percentage change of vasodilation in TM4 homozygous mutant group was significantly lower than those in the wild-type group(P＜0.05).Conclusions:There were several kinds of phenotypic differences between TM4 gene homozygous mutant mice and wild-type mice.