Spyda基因过表达转基因小鼠品系的建立

目的建立Spyda基因过表达转基因小鼠品系用于深入研究Spyda基因的功能。方法利用Gateway技术构建Spyda表达载体,通过DNA显微注射的方式获得Spyda基因过表达的首建鼠。首建鼠与野生型鼠杂交,所得子代中的阳性鼠同窝交配,已传3代以后的阳性小鼠经SYBR Green实时荧光定量PCR方法检测外源基因的起始模板量,通过与杂合子比较来预测小鼠的基因型,再用传统育种方式验证SYBR Green实时荧光定量PCR的结果。结果获得7只首建鼠,通过与野生型鼠杂交获得阳性子代,进行同窝交配,传至第3代,经实时荧光定量PCR方法筛选出4只纯合子的小鼠,经测交验证,其与正常小鼠交配所生的后代均为阳性,证明实时定量PCR的结果是正确的。建立了2个独立的Spyda转基因小鼠纯合子品系。结论建立了稳定遗传的纯合子Spyda基因过表达转基因小鼠品系,可作为工具鼠进一步深入研究Spyda基因的功能。     

Focal and segmental glomerulosclerosis in mice with podocyte-specific expression of mutant alpha-actinin-4

Mutations in the gene encoding alpha-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine alpha-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant alpha-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant alpha-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex.     

Spreading depression-induced expression of c-fos and cyclooxygenase-2 in transgenic mice that overexpress human copper/zinc-superoxide dismutase

Spreading depression (SD) is a wave of sustained depolarization challenging the energy metabolism of cells without causing irreversible damage. SD is a major mechanism of gene induction that takes place in cortical injury, including ischemia. We studied the role of oxygen radicals in SD-induced c-fos and cyclooxygenase-2 (COX-2) induction using transgenic (Tg) mice that overexpress copper/zinc-superoxide dismutase (SOD1). The frequency, amplitude and duration of SD waves were similar in the Tg mice and wild-type littermates. c-fos and COX-2 mRNAs were strongly induced 1 and 4 h after SD. The induction of both genes was slightly but significantly less at 4 h in the Tg mice. The results indicate that even a mild, noninjurious metabolic stimulation increases the concentration of oxygen radicals to the level that contributes to gene expression.     

用于人顶体蛋白酶原基因表达调控和蛋白分选研究的转基因小鼠模型的建立

分别将构建的人顶体蛋白酶原基因（ｈｕｍａｎ ｐｒｏａｃｒｏｓｉｎ,ｈＡＣＲ）５’侧冀序列,第１外显子和编码β－半乳糖苷酶的基因（ｌａｃＺ）的融合基因（载体１）,以及ｈＡＣＲ基因５’侧冀序列与报告基因ｌａｃＺ的融合基因（载体２）,通过显微注射受精卵法制备转基因小鼠,结果如下：载体１：注射８９只卵,移植入９只母鼠单侧卵管中,３只怀孕,产仔９只,存活７只,基因组Ｓｏｕｔｈｅｒｎ杂交检出整合有外源基因的阳     

Expression of the human renin gene in transgenic mice throughout ontogeny

Expression of a human renin genomic DNA clone extending 900 base pairs upstream and 400 base pairs downstream of the gene has been previously examined in adult transgenic mice. In adults, expression of human renin was evident in kidney, reproductive tissues, adrenal gland and lung. Previous studies of mouse and rat renin have demonstrated that kidney renin becomes evident at approximately 15 days of gestation and that expression is localized first to smooth muscle cells of the developing renal arterial tree and becomes progressively restricted to juxtaglomerular cells. As a prelude to performing cell specificity studies to elucidate the pattern of human renin gene expression in the developing kidney, 15.5 and 17.5 days of gestation fetuses and newborns were obtained for expression analysis. Tissues were pooled and expression was examined in kidney, liver, gastrointestinal (GI) tract, lung, heart and brain. The number of transgenic fetuses in each pool was determined by human renin-specific polymerase chain reaction of DNA purified from placenta or tail biopsies. Renal human renin expression was abundant at all three time points. Expression was also evident in the GI tract at 15.5 and 17.5 days of gestation. Interestingly, although no human renin mRNA was evident in lung at 15.5 or 17.5 days of gestation, extremely high levels of human renin mRNA were detected in the newborn lung. Expression of the human renin gene in these tissues was further confirmed by differential primer extension analysis which is capable of differentiating the closely related human and mouse renin messages. These transgenic mice should provide an interesting model to examine the expression and regulation of the human renin gene during kidney development.     

Changes in exercise-induced gene expression in 5'-AMP-activated protein kinase gamma3-null and gamma3 R225Q transgenic mice

5'-AMP-activated protein kinase (AMPK) is important for metabolic sensing. We used AMPKgamma3 mutant-overexpressing Tg-Prkag3(225Q) and AMPKgamma3-knockout Prkag3-/- mice to determine the role of the AMPKgamma3 isoform in exercise-induced metabolic and gene regulatory responses in skeletal muscle. Mice were studied after 2 h swimming or 2.5 h recovery. Exercise increased basal and insulin-stimulated glucose transport, with similar responses among genotypes. In Tg-Prkag3(225Q) mice, acetyl-CoA carboxylase (ACC) phosphorylation was increased and triglyceride content was reduced after exercise, suggesting that this mutation promotes greater reliance on lipid oxidation. In contrast, ACC phosphorylation and triglyceride content was similar between wild-type and Prkag3-/- mice. Expression of genes involved in lipid and glucose metabolism was altered by genetic modification of AMPKgamma3. Expression of lipoprotein lipase 1, carnitine palmitoyl transferase 1b, and 3-hydroxyacyl-CoA dehydrogenase was increased in Tg-Prkag3(225Q) mice, with opposing effects in Prkag3-/- mice after exercise. GLUT4, hexokinase II (HKII), and glycogen synthase mRNA expression was increased in Tg-Prkag3(225Q) mice after exercise. GLUT4 and HKII mRNA expression was increased in wild-type mice and blunted in Prkag3-/- mice after recovery. In conclusion, the Prkag3(225Q) mutation, rather than presence of a functional AMPKgamma3 isoform, directly promotes metabolic and gene regulatory responses along lipid oxidative pathways in skeletal muscle after endurance exercise.     

Overexpression of c-Myc in beta-cells of transgenic mice causes proliferation and apoptosis,downregulation of insulin gene expression,and diabetes

To test the hypothesis that c-Myc plays an important role in beta-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in beta-cells under control of the rat insulin II promoter. F(1) transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of beta-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other beta-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in beta-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, beta-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in beta-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.     

New potent verapamil derivatives that reverse multidrug resistance in human renal carcinoma cells and in transgenic mice expressing the human MDR1 gene.

Multidrug resistance in human renal cell carcinoma is mainly caused by expression of the MDR1 gene and is characterized by a broad spectrum cross resistance to many natural product chemotherapeutic agents. This resistance can be overcome by applying chemosensitizers which inhibit the function of the MDR1 gene product P-glycoprotein. The development of new reversing agents with fewer side effects and a higher potency in modifying resistance is a high priority of research on drug resistance. We have evaluated four new verapamil derivatives on 21 primary human renal cell carcinomas in vitro, and also tested them in an MDR-transgenic mice model. These mice express the human MDR1 gene in their bone marrow cells and measurement of their white blood counts provides a simple, rapid and reliable system to screen for the potency of MDR-reversing agents in vivo. We demonstrate here that all four drugs are effective in reversing multidrug resistance in primary cultures of human renal cell carcinomas when used in combination with vinblastine chemotherapy, and to a lesser extent with doxorubicin or daunomycin chemotherapy. Our in vivo data indicate that two of these reversing agents display low toxicity at high concentrations and are more effective at low, clinically achievable concentrations, than the other two drugs and R-verapamil. These results make the two new drugs attractive candidates to be taken into clinical trials.     

Cytokine gene expression by alloactivated cells in SCID mice

OBJECTIVES: The severe combined immune deficient (SCID) mouse provides a neutral environment to study human immune responses. We therefore tested human gene expression of Interleukin (IL) 2, 4 and 10, interferon gamma (IFNgamma); transforming growth factor beta 1 (TGFbeta1); and CD40 ligand (CD40L) in splenic extracts of SCID mice after engraftment of PBLs from two persons (direct MLR) or one person plus allopeptides (indirect MLR) in the presence or absence of cyclosporin A (CsA) or FK506. METHODS: Cytokine gene expression was detected by RT and quantitative (for IFN-gamma, TGFbeta1 and CD40L) PCR. All cells, allopeptides, CsA (25 mg/kg/day for 7 days) or FK 506 (0.5 mg/kg/day for 7 days) were administered intraperitoneally (IP). RESULTS: In both direct and indirect MLR the numbers of SCID mice expressing the human cytokine genes varied between 33% for IL4 and 100% for IL10, IFN-gamma, TGFbeta1, and CD40L. There was significant interpersonal variation in levels of gene expression. Concomitant CsA or FK506 administration for 7 days did not abrogate early or late (1 week after discontinuation of CsA or FK506) cytokine gene expression in either the direct or indirect MLR, but paradoxically enhanced levels of IFN-gamma, TGFbeta1 and CD40L gene expression in some experiments. CONCLUSIONS: The results explain late rejection after rapid calcineurin inhibitor withdrawal or reduction, and illustrate the potential use of SCID mice as a surrogate model to study graft outcome by determination levels of gene expression and sensitivity to immunosuppressive agents in the in vivo alloresponse.     

Renal gene expression in embryonic and newborn diabetic mice

Several novel genes that are upregulated in diabetic kidneys have been identified. Recently, transforming growth factor beta driven secreted proteins, i.e., connective tissue growth factor and gremlin (bone morphogenetic protein 2), have been identified, and their expression has been correlated with the tissue changes seen in diabetic nephropathy in the adult population. However, there are very few studies reported in the literature that describe the gene expression in the diabetic state during embryonic and neonatal life. It is well known that exposure to glucose or its epimer, i.e., mannose, induces marked dysmorphogenesis of the embryonic metanephros in an organ culture system. These changes are associated with ATP depletion and marked apoptosis, suggesting an oxidant stress in the induction of dysmorphogenesis of the embryonic metanephros. In view of the glucose-induced changes in the fetal metanephros, a diabetic state was induced by the administration of streptozotocin during pregnancy, and newborn mouse kidneys were processed for suppression subtractive hybridization-PCR. In addition, a diabetic state was induced in newborn diabetic mice, and after 1 week their kidneys were harvested and subjected to representational difference analysis of cDNA. Four novel genes with upregulated mRNA expression were identified. They included: (1) a translocase inner mitochondrial membrane 44 that is involved in the ATP-dependent import of preproteins from the cytosol into the mitochondrial matrix; (2) a kidney-specific aldo-keto reductase that utilizes NADPH and NADH as cofactors in the reduction of aromatic aldehydes and aldohexoses; (3) Rap1b, a Ras-related small GTP-binding protein that behaves as a GTPase and cycles between GTP-bound (active) and GDP-bound (inactive) states associated with conformational change, and (4) a fusion protein of ubiquitin polypeptide and ribosomal protein L40 (UbA(52) or ubiquitin/60) that is intimately involved in the ubiquitin-dependent proteasome pathway related to the accelerated degradation of proteins under various stress conditions, such as those seen in patients with cancer and diabetes mellitus.     

成骨细胞特异性过表达h1 calponin转基因小鼠的建立

目的为从整体动物水平研究碱性调宁蛋白（h1-calponin）在骨形成过程中的直接作用，我们构建了成骨细胞中特异性过表达h1—calponin的转基因小鼠模型，并对其表型进行了初步分析。方法构建在成骨细胞特异性启动子（collagen I promoter）驱动下表达h1—calponin的载体（pColl-calponin），纯化DNA片段，再以显微注射的方法将转基因片段导人小鼠受精卵，经移植后得到小鼠。PCR法检测整合到小鼠基因组中的外源基因，提取F1代阳性小鼠原代成骨细胞RNA，RT-PCR检测h1-calponin在小鼠成骨细胞中的表达情况，并观察转基因小鼠表型及体重变化情况。结果PCR检测结果显示1只Go代转基因鼠中检测到阳性信号，RT-PCR显示F1代转基因小鼠成骨细胞中表达h1—calponin较野生对照小鼠明显增加，且转基因小鼠体重与野生小鼠相比有明显降低。结论成功构建了在成骨细胞特异性过表达h1—calponin的转基因小鼠，为深入研究h1—calponin在骨骼发育中的作用提供了良好的动物模型。     

Nephron function in transgenic mice with selective vascular or tubular expression of Angiotensin-converting enzyme

Angiotensin-converting enzyme (ACE) null mice display aberrant renal pathology. Inadequate formation of angiotensin II (Ang II) results in hypotension, loss of fluid homeostasis, lack of urine concentration, and failure to regulate GFR through the tubuloglomerular feedback (TGF) mechanism. For examining the tissue-specific role of ACE in renal structure and regulation of renal filtrate formation, single-nephron GFR, proximal tubular fluid reabsorption, and TGF responsiveness were determined in mice that expressed ACE in only one tissue. Maximum TGF responses in mice that expressed somatic ACE (sACE) in proximal tubule cells (Gs strain) or germinal ACE in the serum (Pg strain) were reduced significantly compared with wild-type (WT) mice. In contrast, TGF responses in mice that expressed sACE in vascular endothelial cells (Ts strain) were not different from control. Single-nephron GFR was reduced in Ts compared with WT mice, but fractional reabsorption and therefore glomerulotubular balance were not distinguishable. BP responses to exogenous Ang I were diminished in Ts, Gs, and Pg mice, whereas those to Ang II were the same in the different strains. Plasma and renal tissue Ang I of all transgenic mouse strains was significantly higher than WT, whereas Ang II levels were generally lower; aldosterone levels were significantly lower than WT in Ts mice but not in the two other transgenic strains. Our results demonstrate that vascular expression of sACE can largely but not completely restore TGF regulation of GFR. Proximal fluid reabsorption in the chronic absence of proximal tubule ACE is normal.     

Reduction in Gal-α1,3-Gal epitope expression in transgenic mice expressing human H-transferase

Abstract: There is now considerable evidence implicating anti-Gal xenoantibodies as a key instigator in the hyperacute rejection of discordant xenografts. As a consequence it is generally held that elimination or reduction of the Gal/anti-Gal component is critical to overcoming hyperacute rejection. We have recently shown that in mice inactivation of the GalT gene by homologous recombination completely eliminates the expression of the Gal-epitope and that hearts from these mice demonstrate prolonged survival when perfused ex vivo with human plasma. Unfortunately this strategy is currently not feasible in pigs because the technology to isolate porcine embryonic stem cells, which are critical for homologous recombination, is not yet available. This study investigates an alternative competition-based transgenic strategy to suppress the level of the Gal epitope by expression of H-transferase (α1,2-fucosyltransferase) an enzyme which has the same substrate specificity (lactobiose) as α1,3-galactosyltransferase. In vitro transfection of murine cells with H-transferase reduced Gal-epitope expression by 80–90%. A similar reduction in Gal expression was observed on PBL and thymocytes from H-transferase transgenic mice. This reduction in Gal epitope expression resulted in a marked reduction in the reactivity of these cells with human serum. In tissues from these mice the reduction in Gal expression was inversely proportional to the endogenous level of Gal. The results of this study support pursuing this strategy as a means to reduce the xenoantigenicity of porcine tissues.