肝特异性表达HCV5′NCR调控荧光素酶质粒的构建及表达

小鼠白蛋白是肝组织特异性表达的蛋白 ,这种特异性是由白蛋白启动子所介导的 .以2 2 35A- 1质粒为模板 ,通过 PCR扩增获得小鼠白蛋白启动子 /增强子基因片段 ,用小鼠白蛋白启动子 /增强子基因片段取代 p HCV- neo4质粒 (含 HCV5′NCR调控荧光素酶基因 )的 CMV启动子 ,构建了一种白蛋白启动子启动转录的 HCV5′NCR调控荧光素酶表达质粒 (p A1 b- HCV) .该质粒能在小鼠肝癌细胞中表达且较小鼠其它癌细胞中表达水平明显增高 ,表明成功地构建了肝特异性表达的 HCV5′NCR调控荧光素酶表达质粒 .该研究为建立肝特异性表达的 HCV5′NCR转基因小鼠模型奠定了基础 ,对评价 HCV特异性反义药物及肝靶向性运载系统的作用具有重要的实际意义     

肝组织特异表达人核受体nr5a2转基因小鼠的构建

人乙肝病毒增强子ⅡB1结合因子（hB1F）系Ftz—F1（NR5A）亚家族的新成员。经基因重组法将人hb1 fcDNA置于小鼠白蛋白增慢子／启动子序列下游构建成肝特异重组载体,通过原核显微注射将该载体导入小鼠受精卵原核,经注射且状态良好的卯回输至假孕母鼠输卯管。产下仔鼠经PCR和Southern blotting鉴定,同时RT—PCR和Western blotting分析转基因的表达。阳性Founder鼠与正常C57鼠交配以建立转基因纯系小鼠,F1代以PCR法鉴定。结果共获得4只PCR鉴定转基因阳性Founder鼠,其中一只同时经Southern blotting鉴定为阳性。RT—PCR和Western blotting结果显示,外源基因在转基因小鼠的肝组织成功表达。遗传学分析表明,转基因已整合入小鼠基因组并可稳定溃传。     

The int-2 gene product acts as an epithelial growth factor in transgenic mice.

The induction of mammary tumors by mouse mammary tumor virus (MMTV) is thought to occur through proviral activation of one or more cellular genes. One of these, int-2, encodes a 27 kd protein which exhibits striking homology to the basic fibroblast growth factor family. To assess directly the role of the int-2 protein in cell proliferation, we have established transgenic mice which carry the int-2 gene driven by the MMTV promoter/enhancer. Expression of the int-2 gene in female transgenic mice results in pronounced mammary gland hyperplasia. Interestingly, expression of the MMTV-int-2 transgene in the prostate gland of male carriers results in a benign, but dramatic, epithelial hyperplasia similar to benign prostatic hypertrophy (BPH), a common but poorly understood disorder in human populations. Together, these results indicate that the int-2 product can act as a potent growth factor in these epithelial tissues.     

T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.

A hybrid mouse major urinary protein (MUP)/SV40 T antigen gene was microinjected into fertilized mouse embryos and the resulting transgenic mice analyzed for the regulated expression of the transgene. Available evidence indicates that the MUP gene used for the hybrid gene construct is expressed in both male and female liver and possibly mammary gland. Three different transgenic lines exhibited a consistent pattern of tissue specific expression of the transgene. As a consequence of transgene expression and T antigen synthesis in the liver, both male and female transgenic animals developed liver hyperplasia and tumors. Transgene expression and liver hyperplasia commenced at approximately 2-4 weeks of age, the same time that MUP gene expression is first detected in the liver. The expression of the transgene resulted in an immediate strong suppression of liver MUP mRNA levels but had relatively little effect on other liver specific mRNAs. From 4 to 8 weeks, the liver increased several fold in size, relative to non-transgenic littermates. Definitive tumor nodules were not apparent until 8-10 weeks. The transgene was also consistently found to be expressed in the skin sebaceous glands and the preputial gland, a modified sebaceous gland. The expression of the transgene in the skin sebaceous glands is consistent with the presence of MUP mRNA in the skin and a putative role for MUPs in the transport and excretion of small molecules. Occasional expression of the transgene in other tissues (kidney and mammary connective tissues) was also noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

φC31 integrase and liver-specific regulatory elements confer high-level,long-term expression of firefly luciferase in mouse liver

To improve the efficiency of expression of reporter transgenes delivered by hydrodynamic injection, we generated expression cassettes carrying different liver-specific regulatory elements using the firefly luciferase gene as a reporter. From our studies the human alpha-antitrypsin promoter together with the apolipoprotein E/C-I and albumin enhancer was the combination of choice for prolonged transgene expression, but reporter gene expression in vivo lasted for no more than 7 weeks. Subsequently, phage φC31 integrase was introduced as a potential tool to improve further the efficiency of expression of the delivered transgene. Long-term transgene expression in vivo was achieved by specific integration of the target gene into mouse livers. This study demonstrates the use of a combination of phage φC31 integrase and liver-specific regulatory elements for generation of transgenic mice.     

In vitro Cre/loxP system in cells from developing gonads: investigation of the Sry promoter

There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.     

Neonatal bleeding in transgenic mice expressing urokinase-type plasminogen activator.

Spontaneous intestinal and intra-abdominal bleeding was observed in a high percentage of newborn transgenic mice carrying the murine urokinase-type plasminogen activator (uPA) gene linked to the albumin enhancer/promoter. These hemorrhagic events were directly related to transgene expression in the liver and the development of high plasma uPA levels. Two lines were established from surviving founder mice that displayed multigenerational transmission of the bleeding phenotype. Fatal hemorrhaging developed between 3 and 84 hr after birth in about half of the transgenic offspring of these lines; transgenic pups that did not bleed nevertheless passed the phenotype to their young. The phenotypic variability could not be explained by differences in transgene expression. All transgenic neonates were severely hypofibrinogenemic and displayed loss of clotting function that extended beyond the risk period for bleeding. These mice provide a means of studying the pathophysiology of plasminogen hyperactivation and evaluating therapeutic protocols designed to prevent bleeding.     

Tissue-specific and hormonally regulated expression of a rat alpha 2u globulin gene in transgenic mice.

To investigate the tissue-specific and hormonal regulation of the rat alpha 2u globulin gene family, we introduced one cloned member of the gene family into the mouse germ line and studied its expression in the resulting transgenic mice. Alpha 2u globulingene 207 was microinjected on a 7-kilobase DNA fragment, and four transgenic lines were analyzed. The transgene was expressed at very high levels, specifically in the liver and the preputial gland of adult male mice. The expression in male liver was first detected at puberty, and no expression was detected in female transgenic mice. This pattern of expression is similar to the expression of endogenous alpha 2u globulin genes in the rat but differs from the expression of the homologous mouse major urinary protein (MUP) gene family in that MUPs are synthesized in female liver and not in the male preputial gland. We conclude that these differences between rat alpha 2u globulin and mouse MUP gene expression are due to evolutionary differences in cis-acting regulatory elements. The expression of the alpha 2u globulin transgene in the liver was abolished by castration and fully restored after testosterone replacement. The expression could also be induced in the livers of female mice by treatment with either testosterone or dexamethasone, following ovariectomy and adrenalectomy. Therefore, the cis-acting elements responsible for regulation by these two hormones, as well as those responsible for tissue-specific expression, are closely linked to the alpha 2u globulin gene.     

Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.     

Fkbp8: novel isoforms, genomic organization, and characterization of a forebrain promoter in transgenic mice

The immunophilin homolog FKBP8 has been implicated in the regulation of apoptosis. Here we show that the 38-kDa form of FKBP8 (FKBP38) derives from a truncated ORF. The extended FKBP8 ORFs are 46 and 44 kDa in mouse and 45 kDa in human. Although the genomic organization of mouse and human FKBP8 is evolutionarily conserved, additional first exons are encoded by the murine locus. A 4.4-kb murine Fkbp8 gene fragment, containing a GC-rich potential promoter, directed expression of a LacZ reporter gene to forebrain neurons in transgenic mice. Expression of the transgene was observed in CA1 pyramidal neurons of the hippocampus in transgenic mice from three lines. One transgenic founder mouse exhibited widespread forebrain expression of the LacZ transgene that resembles the pattern for the endogenous Fkbp8 gene. Thus promoter/enhancer elements for forebrain expression are located around the first exons of the mouse Fkbp8 gene.     

A 5-kilobase pair promoter fragment of the murine epididymal retinoic acid-binding protein gene drives the tissue-specific, cell-specific, and androgen-regulated expression of a foreign gene in the epididymis of transgenic mice

The murine epididymis synthesizes and secretes a retinoic acid-binding protein (mE-RABP) that belongs to the lipocalin superfamily. The gene encoding mE-RABP is specifically expressed in the mouse mid/distal caput epididymidis under androgen control. In transgenic mice, a 5-kilobase pair (kb) promoter fragment, but not a 0.6-kb fragment, of the mE-RABP gene driving the chloramphenicol acetyltransferase (CAT) reporter gene restricted high level of transgene expression to the caput epididymidis. No transgene expression was detected in any other male or female tissues. Immunolocalization of the CAT protein and in situ hybridization of the corresponding CAT mRNA indicated that transgene expression occurred in the principal cells of the mid/distal caput epididymidis, thereby mimicking the spatial endogenous mE-RABP gene expression. Transgene and mE-RABP gene expression was detected from 30 days and progressively increased until 60 days of age. Castration, efferent duct ligation, and hormone replacement studies demonstrated that transgene expression was specifically regulated by androgen but not by any other testicular factors. Altogether, our results demonstrate that the 5-kb promoter fragment of the mE-RABP gene contains all of the information required for the hormonal regulation and the spatial and temporal expression of the mE-RABP gene in the epididymis.     

Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice.

Regulatory regions of the mouse muscle creatine kinase (MCK) gene, previously discovered by analysis in cultured muscle cells, were analyzed in transgenic mice. The 206-bp MCK enhancer at nt-1256 was required for high-level expression of MCK-chloramphenicol acetyltransferase fusion genes in skeletal and cardiac muscle; however, unlike its behavior in cell culture, inclusion of the 1-kb region of DNA between the enhancer and the basal promoter produced a 100-fold increase in skeletal muscle activity. Analysis of enhancer control elements also indicated major differences between their properties in transgenic muscles and in cultured muscle cells. Transgenes in which the enhancer right E box or CArG element were mutated exhibited expression levels that were indistinguishable from the wild-type transgene. Mutation of three conserved E boxes in the MCK 1,256-bp 5' region also had no effect on transgene expression in thigh skeletal muscle expression. All these mutations significantly reduced activity in cultured skeletal myocytes. However, the enhancer AT-rich element at nt - 1195 was critical for expression in transgenic skeletal muscle. Mutation of this site reduced skeletal muscle expression to the same level as transgenes lacking the 206-bp enhancer, although mutation of the AT-rich site did not affect cardiac muscle expression. These results demonstrate clear differences between the activity of MCK regulatory regions in cultured muscles cells and in whole adult transgenic muscle. This suggests that there are alternative mechanism of regulating the MCK gene in skeletal and cardiac muscle under different physiological states.     

Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter

To investigate the ability of 1.8 kb or 3.1 kb bovine beta-casein promoter sequences for the expression regulation of transgene in vivo, transgenic mice were produced with human type II collagen gene fused to 1.8 kb and 3.1 kb of bovine beta-casein promoter by DNA microinjection. Five and three transgenic founder mice were produced using transgene constructs with 1.8 kb and 3.1 kb of bovine beta-casein promoters respectively. Founder mice were outbred with the wild type to produce F1 and F2 progenies. Total RNAs were extracted from four tissues (mammary gland, liver, kidney, and muscle) of female F1 transgenic mice of each transgenic line following parturition. RT-PCR and Northern blot analysis revealed that the expression level of transgene was variable among the transgenic lines, but transgenic mice containing 1.8 kb of promoter sequences exhibited more leaky expression of transgene in other tissues compared to those with 3.1 kb promoter. Moreover, Western blot analysis of transgenic mouse milk showed that human type II collagen proteins secreted into the milk of lactating transgenic mice contained 1.8 kb and 3.1 kb of bovine beta-casein promoter. These results suggest that promoter sequences of 3.1 kb bovine beta-casein gene can be used for induction of mammary gland-specific expression of transgenes in transgenic animals.     

Influence of a nonfragile FHIT transgene on murine tumor susceptibility

FHIT, at a constitutively active chromosome fragile site, is often a target of chromosomal aberrations and deletion in a large fraction of human tumors. Inactivation of murine Fhit allelessignificantly increases susceptibility of mice to spontaneous and carcinogen-induced tumorigenesis. In this study, transgenic mice, carrying a human FHIT cDNA under control of the endogenous promoter, were produced to determine the effect of Fhit expression, from a nonfragile cDNA transgene outside the fragile region, on carcinogen-induced tumor susceptibility of wildtype and Fhit heterozygous mice. Mice received sufficient oral doses of N-nitrosomethybenzylamine (NMBA) to cause forestomach tumors in >80% of nontransgenic control mice. Although the level of expression of the FHIT transgene in the recombinant mouse strains was much lower than the level of endogenous Fhit expression, the tumor burden in NMBA-treated male transgenic mice was significantly reduced, while female transgenic mice were not protected. To determine if the difference in protection could be due to differences in epigenetic changes at the transgene loci in male versus female mice, we examined expression, hypermethylation and induced re-expression of FHIT transgenes in male and female mice or cells derived from them. The transgene was methylated in male and female mice and in cell lines established from male and female transgenic kidneys, the FHIT locus was both hypermethylated and deacetylated. It is likely that the FHIT transgene is more tightly silenced in female transgenic mice, leading to a lack of protection from tumor induction.