Objective To establish a double reporter transgenic mice for monitoring Cre recombinase activity. Methods ZAP DNA fragment with lacZ and human alkaline phosphatase (hAP) gene was microinjected into the male pronucleus of 554 fertilized eggs from C57BL/6 mice. The founder mice and their progeny were screened for integration of transgene into the mouse genome by PCR and Southern blotting. The expression of lacZ transgene at early embryos from F1 generation mice was analyzed by X gal staining. Results A total of 398 survival ZAP DNA injected fertilized eggs were transfered to the oviducts of 21 pseudopregnant recipient mice. Of the 21 recipient mice, 13 became pregnancy and gave birth to 68 offspring mice. The zygote survival rate and birth rate were 71% (398/554) and 17% (68/398), respectively. Of the 68 offspring mice, 9 mice (5 males and 4 females) were identified by PCR and Southern blot analysis. Total integration rate and efficiency of transgene was 13% (9/68) and 1.6% (9/554), respectively. Nine mice as the founders were back crossed to set up F1 generation with other inbred C57BL/6 mice. Out of 9 transgenic mice, transmission of reporter gene in F1 offspring mice followed Mendelian rules, but the expression of lacZ protein was detected at the early embryonic stage (13.5 days postcoitum) in only 3 mice. Conclusion A double reporter transgenic mice for monitoring Cre recombinase activity is established.

Objective To establish the replicated-deficient recombinant adenovirus-mediated thymidine kinase/acyclovir (Adtk/ACV) system and to evaluate its suicide effect on rat C6 brain gliomas in vitro and in vivo.Methods The plasmid pAdtk and pJM17 were co-infected into 293 cells (adenovector packaging cells) and the results were identified by polymerase chain reaction (PCR) assay. After the glioma C6 cells were transduced by Adtk at different multiplicity of infection (MOI) and exposed to different concentrations of ACV or gancyclovir (GCV), the cell survival curves were studied, and the cell surface was observed with scanning electronic microscopy (SEM). C6 gliomas in vivo at different inoculation days were injected with Adtk intratumorally and ACV intraperitoneally daily, and the survival duration and histologic changes of the rats were observed.Results The infectious Adtk virions had a suicide effect which was enhanced with the increase in MOIs of Adtk and ACV doses along with bystander effect. Under scanning electronic microscope, special pathologic changes were observed. ACV had a similar effect as GCV but a higher dose was used. The survival duration in day 3, day 6 and day 8 groups exceeded 90 days, and the rats in day 10 group survived 28.5±4.6 days, but the survival duration in untreated C6 group and AdLacZ/ACV (adenovirus-mediated LacZ/ACV) treated group were 16.8±3.1 and 14.0±2.2 days respectively. Conclusion Adtk/ACV system can effectively kill the rat brain gliomas in vitro and in vivo.

Objective To establish the polymorphism of DXS102 locus from Xq26.3-27.1 in Chinese population for the gene diagnosis in Hemophilia B family.Methods DNA was extracted from blood samples obtained from Shanghai unrelated volunteer donors with phenol-chloroform method. A total of 23 × chromosomes (154 from females, 80 from males) were studied. A hemophilia B family in which a hemophilia B patient has received gene therapy was analyzed. The polymorphism of DXS102 locus in Chinese population was determined with amplified fragment length polymorphisms assay (Amp-FLP), denaturing polyacrylamide gel electrophoresis, silver stain detection. Short tandem repeats (STRs) linkage analysis was used to conduct gene diagnosis in hemophilia B family.Results Eight alleles were found at DXS102 locus, of which two alleles were first reported. The repeated number of AC dinucleotide ranges from 13 to 21. And the values of the observed heterozygosity, calculated heterozygosity and polymorphism information content(PIC) were 0.87, 0.80, 0.80 respectively. It was also found that the difference of the allele frequencies of DXS102 in Chinese and European populations was significant. By using the linkage analysis of the DXS102 locus, a family with a hemophilia B patient receiving gene therapy in 1994 was analyzed and meanwhile a carrier in that family was then detected.Conclusions The polymorphism of DXS102 locus reveals significant difference between Chinese and European populations. DXS102 locus can be used as a promising marker for gene diagnosis in hemophilia B family.

OBJECTIVETo investigate the association of two single nucleotide polymorphisms (SNPs) of beta 2-adrenoceptor (beta 2-AR) gene with hypertension in elderly patients.

METHODSThe study samples were collected from unrelated Chinese Han population of Dabie Mountain in Anhui province. Eighty-six elderly patients with hypertension and 43 controls were selected. Genotypes of +1053 and +1239 SNPs were typed by polymerase chain reaction-restriction fragment length polymorphism.

RESULTSThe frequencies of the two SNPs complied well with the Hardy-Weinberg equilibrium in normal group. The distribution of genotypes AA, GA,GG of the SNP at locus +1239 in moderate and severe hypertension group was significantly different from that in normal group (chi square=8.67, P<0.05). There were evident differences in the frequencies of alleles of the two groups (chi square=4.02, P<0.05). No significant difference was observed in the distribution of genotypes of the SNP at locus +1053 between the two groups.

CONCLUSIONThese data indicate that the SNP at locus +1239 of beta 2-AR gene is associated with hypertension in elderly patients.

Aged ; Aged, 80 and over ; Alleles ; DNA ; genetics ; Female ; Gene Frequency ; Genotype ; Humans ; Hypertension ; genetics ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Receptors, Adrenergic, beta-2 ; genetics

Objective　The study was conducted to investigate single nucleotide polymorphism(SNP) in beta2-adrenoceptor(β2-AR) gene and the distribution of these identified SNPs in Chinese Han ethnic group.Methods　β2-AR gene was sequenced to detect SNPs by fluorescent labeling automatic sequencing method in 80 unrelated samples from territory of Dabie Mountain in Anhui province.Results A total of 8 SNPs were identified in length of 3.8 kb, including 5 SNPs in code region, 3 SNPs in regulatory region. Although the variations, -468C to G, -367T to C, -47C to T,-20T to C,+79C to G,+100G to A,+491C to T,+1098T to C have been identified in other ethnic groups, they have not been found in our study. The allele distribution of SNPs is in good unity with the Hardy-Weinberg equilibrium.Conclusion　The distribution of SNPs in β2-AR gene is not equable and the SNPs in different ethnic groups differ greatly. The allele distribution of SNPs conforms well to the Hardy-Weinberg equilibrium.

Objective　To study whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH-1) and multidrug resistance gene (MDR1) have increases resistance to 4-Hydroperoxycyclo-phosphamide (4-HC) and P-glycoprotein effluxed drugs. Methods　A bicistronic retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed and used to transfect the packaging cell lines GP+E86 and PA317 by LipofectAMINE method, using the medium containing VCR and 4-HC agents for cloning selection and ping-ponging supernatant infection between the ecotropic producer clone and the amphotropic producer clone, we obtained high titer amphotropic PA317 producing cells with high titers up to 5.6×105 CFU/ml. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human ALDH-1 and MDR1cDNA under the stimulation of hemopoietic growth factors. Results　Bicistronic retroviral vector construction was verified by restriction endonuclease analysis. Polymerase chain reaction (PCR), reverse transcription (RT)-PCR, Southern blot, Northern blot, fluorescenceactivated cell sorting (FACS) method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analyses showed that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4-fold stronger resistance to 4-HC and 5.5 to 7.2-fold P-glycoprotein effluxed drug than untransduced cells. Conclusion　The bicistronic retroviral vector-mediated transfer of two different types of drug resistance genes into human cord blood CD34+ cells and co-expression provided an experimental foundation for improving combination chemotherapy tolerance in tumor clinical trial.