Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F)

AIM: Human hepatitis B virus enhancer II B1 binding factor (hBIF) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hBIF transgenic mouse model to promote the functional study of hB1F.METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice.The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis.RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages.Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably.CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

Establishment of transgenic mice carrying gene encoding human zinc finger protein 191

AIM:Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Krueppel-like transcription factor, which might be relevant to many diseases such as liver cancer,neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF191 transgenic mouse model,which would promote the functional study of ZNF191.METHODS:Transgene fragments were microinjected into fertilized eggs of mice.The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice.The offsprings were identified by PCR and Southern blot analysis.ZNF191 gene expression was analyzed by RT-PCR.Transgenic founder mice were used to establish transgenic mouse lineages.The first generation (F1) and the second generation (F2) mice were identified by PCR analysis.Ten-week transgenic mice were used for pathological examination.RESULTS: Four mice were identified as carrying copies of ZNF191 gene.The results of RT-PCR showed that ZNF191 gene was expressed in the liver,testis and brain in one of the transgenic mouse lineages.Genetic analysis of transgenic mice demonstrated that ZNF191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice.CONCLUSION:ZNF191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

Improved immunogenicity of a tuberculosis DNA vaccine encoding ESAT6 by DNA priming and protein boosting

The study evaluated the immune response elicited by a DNA vaccine encoding EbAT6 protein of Mycobacterium tuberculosis by DNA prime-protein boost protocol. BALB/c mice were respectively vaccinated with plasmid DNA encoding ESAT6 protein, with ESAT6 protein in IFA adjuvant, or a combined DNA prime-protein boost regimen. While DNA immunization induced The-polarized immune response, protein-in-adjuvant vaccination elicited a Thz-dominant response. When animals were primed with DNA and boost with protein, both antibodies and Th-cell proliferative response were significantly enhanced. Moreover, production of The-type cytokine (IFN-gamma) was increased significantly by DNA priming-protein boosting.

Allelotyping for loss of heterozygosity on chromosome 18 in gastric cancer

AIM: To investigate the association between loss of heterozygosity (LOH) on chromosome 18 and sporadic gastric cancer.METHODS: Multiplex PCR was used to screen 14 highly polymorphic microsatellite markers on chromosome 18 in 45 cases of primary gastric cancer. PCR products were separated on polyacrylamide gels and the electrophoresis maps were analyzed with Genescan and Genotyper.RESULTS: The LOH frequencies in gastric cancer at all 14 markers ranged from 10% to 58%. Eleven markers were found with over 20% LOH frequencies, in which 9 markers located in 18q, and 2 markers in 18p. Two overlapping deleted regions were identified: R1 between D18S61-D18S1161 at 18q22 (9cM) with 24% LOH frequency; R2 between D18S462D18570 at 18q22-23(6cM) with 32% LOH frequency.CONCLUSION: LOH of chromosome 18 (18q and 18p) may be involved in gastric tumorigenesis. Two overlapping deleted fragments suggested that there might be unidentified tumor suppressor genes in those two regions.

Gene expression profile in liver of hB1F transgenic mice

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hBIF transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of bhe differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hBIF were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hBIF transgene may cause changes of gene expression profiles in the liver of transgenic mice.

一种筛查中国人遗传性耳聋和氨基糖苷耳毒性易感人群的分子遗传学方法(英文)

Objective: To develop a molecular screening test for genetic defects on hearing loss related genes has significant impacts on early identification of hereditary hearing loss and genetic susceptibility to aminoglycoside ototoxicity. Early identification of pre-lingual hearing loss is very important for patient’s language development, academic achievement, and social skill. Two common mutations, the 235delC in GJB2 gene and the mutation A1555G in mitochondrial DNA, are included in the newly developed screening panel for Chinese population. Methods: A molecular genetic assay, based on fluorescent labeled multiplex PCR and automatic DNA fragment analyzing techniques, was developed to detect both mutations simultaneously. Results: This assay was able to detect both mutations from patient’s samples, and pooled DNA tests, as well as suitable to detect mutation from the DNA extracted from dried blood spot and buccal swab. Conclusion: This assay could be a useful tool for newborn screening and carrier screening for the hereditary hearing loss for the Chinese population.

Application of Short Tandem Repeat in Prenatal Diagnosis for Phenmylketonuria during the First Trimester

Objective : To find a simple and rapid way far the prenatal diagnosis of phenyUce-tonuria (PKU) during the first trimester in order to prevent inborn PKU patients as early as possible. Methods :DNA was extracted respectively from the Mood sampleps of 9 families' members and chori-onic tissues of 9 embryoes by cliorionic vittus sampling (CVS). The independent short tandem repeat (STR) alleles of members in 9 families with classic form of PKU were analyzed and prenatal diagnosis were conducted using polymerase chain reaction (PCR) together with denaturing gradient gel elec-trophoresis(DGGE)and silver dyeing. Results-.We identified 1 embryo with PKU, 2 normal individuals and 5 carriers among 9 subjects. Conclusion: Prenatal diagnosis for PKU by STR is available in the first trimester. This procedure was promising and would be widely used in Chinese population.

Non-fusion expression in Escherichia coli, purification, and characterization of a novel Ca^2＋- and phospho-lipid-binding protein annexin B1

Annexin B1 is a novel member of the annexin family of Ca^2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). Alter induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture.

Construction of fine SNP haplotypes and haplotype blocks in 5 genes in the centromere of chromosome 15 in Chinese Han subjects

Recent advances have shown that the majorityof the nucleotide variation in human genome is single nucleo-tide polymorphisms (SNPs). Using SNPs each chromosomecan be divided into different haplotype blocks, and there arelimited common haplotypes in each block. This provides apowerful approach for whole genome scan for disease-asso-ciated genes/variants. However, most data available todayare based on the large-scale genomic analyses, data concern-ing individual genes for fine mapping with high density SNPsare relatively lacking. We have sequenced 7 genes and theirflanking regions, identified 34 novel SNPs, constructed highdensity SNP haplotypes and haplotype blocks in 5 genes inthe centromeric region of chromosome 15 in I00 ChineseHart subjects. Our results show that there is a great hetero-geneity in the haplotypes and haplotype block structureswithin and between these genes, which are in close physicalproximity. Data obtained in this study provide a useful toolfor candidate gene approach at the fine scale for identifyingdisease contributing variants in the genes/regions.

Establishment of widely expressed hb1f transgenic mouse lineage and its morphologic analysis

Human nuclear receptor hB1F is a novel member of the fushi tarazu factor Ⅰ subfamily of nuclear receptor superfamily. The studies about its homologous genes indicate that hB1F may play a key role in regulating the metabolic homeostasis of cholesterol. After obtaining the founder mice carrying the trausgeue of hblf by microiujectiou, each founder was mated to normal C57 mouse and the positive F1 by PCR identification of the same founder were iutercrossed within sisters and brothers to establish the trausgeuic mouse lineage. The results of F1, F2 and offspring of test cross identification showed that the widely expressed hb1f trausgeuic mouse lineage was established successfully in this study. The tissue morphology of the trausgeuic lineage was also analyzed preliminarily.

RESTRICTIVE FRAGMENT LENGTH POLYMORPHISMS OF MARKERS 3'HVR AND 24-1 AND THEIR APPLICATIONS IN GENE DIAGNOSIS OF ADULT POLYCYSTIC DISEASE IN CHINESE

Adult polycystic kidney disease was diagnosed with pedi-gree analysis using polymorphic DNA markers linked to thedisease gene in 5 families. The markers were thoroughly charac-terized in the population. We noted two important markers,3’HVR and 24-1, which flank the disease gene PKD1 in nor-mal Chinese. Southern hybridization was used, and numerous3’HVR / Pvull polymorphic allelic bands varied from 1.4 kb to8.0 kb with a positive skew distribution. They were divided into11 groups with an interval of 0.6 kb and a heterozygosity ratcof 0.86. Allele frequencies were observed. When the probe24-1 was hybridized with genomic DNA digested by TaqI, twoalleles, B1 and B2, were detected with frequencies of 0.76 and0.24, respectively. B1 was 3.8 kb in size and B2 was composedof 2 bands, 1.5 kb and 1.3 kb. The polymorphism informationcontent was found to be 0.297. Using these two markers forlinkage analyses of 5 affected families, we confirmed thelinkage between 3’HVR and the disease gene PKDI and madepresymptomatic