乙型肝炎病毒颗粒性抗原转基因番茄植株的构建及鉴定

目的 构建乙型肝炎病毒(HBV)颗粒性抗原转基因番茄植株。方法 将乙型肝炎病毒preS／S基因插入到HBc的第73～94aa处(棘突尖部)，再把整个复合基因克隆到植物表达载体pBIN438(含35s起动子)中，通过液氮冻融法转化根瘤农杆菌ehal05，采用叶盘法转化番茄。结果 得到一批转基因番茄植株，经PCR、PCR-Southern blot和Souhtem blot证实HBc-HBs基因已整合到番茄基因组中；经Western blot证实HBc-HBs能在番茄中有效表达。结论 成功构建了乙肝颗粒性抗原基因的转基因番茄植株，为下一步进行该番茄疫苗株的效果评价奠定了基础。     

NUP98-PMX1转基因小鼠发生髓系白血病样表型

目的　在整体动物水平研究NUP98 PMX1融合蛋白的致白血病作用。方法　采用分子克隆技术构建含有NUP98 PMX1的转基因质粒 ,显微注射法建立转NUP98 PMX1基因的小鼠 ,以PCR、RT PCR方法检测融合基因的表达 ;用流式细胞仪检测骨髓细胞表型。结果　转染NUP98 PMX1融合基因的NIH3T3细胞生长加快 ,软琼脂上可形成集落 ,并使裸鼠致瘤。出现病态的 8只NUP98 PMX1转基因小鼠中有 4只发生粒细胞白血病样表型 ,其中 3只表现为人类慢性髓系白血病样表型。结论　NUP98 PMX1融合基因具有致瘤性 ,其表达在髓系白血病的发生中起着极为重要的作用。     

单克隆抗体诱发乙肝转基因小鼠肝组织损伤

目的：诱导整合有乙型肝炎病毒（HBV）全基因组、无病理学反应的转基因小鼠产生病理学改变，建立研究急性肝炎发生机制及治疗药物筛选的转基因动物模型。方法：采用尾静脉注射方法将3种HBV单克隆抗体HBsAb、HBeAb、HBcAb分别或混合导入整合有HBV全基因组的转基因小鼠体内，使之与体内的抗原结合产生免疫学反应，随后进行血清学及常规病理学的研究。结果：3种单克隆抗体单独或混合处理均引起HBV转基因小鼠肝细胞的病理学损伤，以HBsAb及HBsAb HBcAb混合处理组损伤较为严重；但小鼠血清转氨酶基本表现正常。结论：体液免疫反应可以诱导HBV转基因小鼠组织细胞发生损伤，并产生与急性肝炎相似症状的病理学改变，HBsAb及HBsAb HBcAb处理的HBV转基因小鼠可以作为研究急性肝炎发生机制的动物模型。     

用显微注射法建立转bcl-x_l基因小鼠

建立转bcl xl基因小鼠 ,继而传代建系 ,用于缺血性脑血管疾病的研究。采用显微注射法 ,将人bcl xl基因注入昆明小白鼠受精卵获得子代鼠 ,然后作PCR ,Southern blot ,mRNA ,Western blot检测以获得阳性鼠。实验中注射受精卵 16 5 4枚 ,移植卵数 14 4 8枚 ,受体鼠 4 9只 ,怀孕鼠 7只 ,子代鼠 13只 ,整合数 4只并均有表达 ,植入受精卵的存活率 83% ,受精卵的总存活率 0 .78% ,移植鼠的怀孕率 14 .3% ,整合效率 30 .7% ,总有效率为 0 .2 4 %。初步获得了转bcl xl基因小鼠     

cDNA sequence of neuroendocrine protein 7B2 expressed in beta cell tumors of transgenic mice

The cDNA for a widely distributed neuroendocrine protein called 7B2 has been cloned from beta cell tumors of transgenic mice and sequenced. As deduced from the cDNA sequence, 7B2 is a secretory protein of 186 amino acids, nearly identical to its human and porcine homologs. The presence of several pairs of basic residues in the carboxyl terminal portion of the protein suggests that 7B2 can undergo proteolytic maturation in secretory granules and thus generate potential bioactive peptides. 7B2 mRNA is about 1.5 kilobase long and is apparently transcribed from a single gene per haploid genome. The use of tissue-specific promoters to express oncogenes in rare cell types of transgenic mice is a powerful tool for immortalization and expansion of these cells, and it facilitates the isolation and the study of rare proteins such as 7B2.     

转基因中国仓鼠卵细胞中单胺囊泡转运体抗毒性作用的研究

目的:研究转基因中国仓鼠卵(CHO)细胞中单胺囊泡转运体(VMAT_2)的抗毒性作用。方法:利用转PC12细胞基因到CHO细胞中形成的转基因CHO细胞(cDNA-CHO),采用MTT比色法检测1-甲基-4-苯基吡啶离子(MPP~+)对CHO细胞野生株(wtCHO)和cDNA-CHO细胞的毒性作用,并观察利血平——VMAT_2的特异性阻滞剂对MPP~+毒性作用的影响。结果:在MPP~+ 0.5mmol/L以上浓度cDNA-CHO细胞对MPP~+敏感性比wtCHO低得多;cDNA-CHO和wtCHO对鱼藤酮(rotenon)的敏感性无显著差异;加入利血平后,上述保护作用消失,cD-NA-CHO对MPP~+敏感性与wtCHO细胞无差异,而单独予以wtCHO细胞利血平则不能改变它对低浓度MPP~+的敏感性。结论:此保护机制是由转基因细胞中VMAT_2引起的,VMAT_2在转基因的非神经细胞系(CHO细胞系)中也能将MPP~+转运至囊泡内,从而保护细胞,同时也提示PC12细胞内具有抗毒性作用的成分。     

HPA axis dysregulation in mice overexpressing corticotropin releasing hormone.

BACKGROUND: Hypersecretion of corticotropin-releasing hormone (CRH) in the brain has been implicated in stress-related human pathologies. We developed a transgenic mouse line overexpressing CRH (CRH-OE) exclusively in neural tissues to assess the effect of long-term CRH overproduction on regulation of the hypothalamic-pituitary-adrenal (HPA) axis. METHODS: Male transgenic CRH-OE(2122) mice on a C57BL/6J background were used. Littermate wildtype mice served as control animals. Basal plasma corticotropin and corticosterone concentrations were measured, and adrenal gland weight was determined. A dexamethasone suppression test measured the effects of long-term CRH hypersecretion on negative feedback control. Additionally, we measured plasma corticosterone concentrations in reaction to stress. RESULTS: CRH-OE(2122) mice showed elevated basal plasma corticosterone concentrations, hypertrophy of the adrenal gland, and dexamethasone nonsuppression. Basal plasma ACTH concentrations of wildtype and CRH-OE(2122) mice did not differ significantly. In reaction to stress, CRH-OE(2122) mice showed a normal corticosterone response. CONCLUSIONS: The HPA axis abnormalities observed in CRH-OE(2122) mice suggest that long-term hypersecretion of CRH in the brain can be a main cause of HPA axis dysregulation. The alterations in HPA axis regulation are reminiscent of changes reported in major depressive disorder. As such, these CRH -OE(2122) mice may model the neuroendocrine changes observed in major depressive disorder.     

人bcl-xl转基因小鼠外源基因的复制及传代的稳定性观察

显微注射法建立转bcl-xl基因小鼠，将PCR及Southern-blot检测阳性的小鼠与正常鼠酱并传代，然后检测子代中外源基因的遗传 情况。结果发现：10号小鼠传代过程中PCR阳性率保持稳定，符合孟德尔遗传规律，外源基因能够稳定遗传 。6号小鼠PCR阳性率呈轻微下降趋势，但能够保持相对稳定遗传 。13号和5号小鼠PCR阳性率均呈现明显的下降趋势。该二小鼠存在外源基因遗传丢失现象。结果提示：获得了能够稳定遗传 的转基因鼠系。     

Podocyte changes upon induction of albuminuria in Thy-1.1 transgenic mice.

BACKGROUND: Thy-1.1 transgenic mice, characterized by ectopic expression of the Thy-1.1 protein on podocytes, spontaneously develop proteinuria and focal glomerulosclerosis (FGS). Injection of a monoclonal antibody (mAb) directed against the Thy-1.1 protein in young transgenic mice induces a massive albuminuria that is followed by an accelerated FGS within 3 weeks. This albuminuria is complement and leukocyte independent. The time course of proteinuria, the pathogenesis of the acute proteinuria and the dose dependency of FGS are unknown. METHODS: Albuminuria was measured in Thy-1.1 transgenic mice after injection of different doses of anti-Thy-1.1 mAb and at different time points within the first 24 h after injection. Podocytic foot processes and slit pore diameter were quantitated by electron microscopy. Changes in expression of slit pore constituents (podocin, CD2AP, nephrin and ZO-1), cytoskeleton-associated proteins (actin, alpha-actinin, ezrin and synaptopodin), the GDH-podocyte adhesion molecules alpha(3)-integrin, and heparan sulfate were studied by immunofluorescence. FGS was scored by light microscopy at 3 weeks after induction of albuminuria. RESULTS: Albuminuria in Thy-1.1 transgenic mice was observed within 10 min after anti-Thy-1.1 mAb injection. This rapid development of albuminuria was accompanied by a reduction in number of podocytic foot processes from 20.0 +/- 0.7/10 microm glomerular basement membrane (GBM) in saline-treated transgenic mice to 8.0 +/- 0.5 and 2.2 +/- 0.2 in anti-Thy-1.1-treated mice, at 10 min and 8 h after treatment, respectively. In addition, we observed a significant decrease in width of remaining slit pores, from 32.7 +/- 1.1 to 26.8 +/- 1.4 nm at 10 min after mAb injection. By immunofluorescence, we did not observe major changes in the expression pattern of any of the proteins studied. There was no correlation between the injected dose of the anti-Thy-1.1 mAb and the acute albuminuria. In contrast, the percentage of FGS at 3 weeks correlated with the dose, and a significant correlation between the percentage of FGS and the time-averaged albuminuria over the 3 week study period (P < 0.001) was found. CONCLUSION: Injection of mAb directed against the Thy-1.1 protein, in young non-albuminuric Thy-1.1 transgenic mice, induced an acute albuminuria within 10 min, which was accompanied by foot process effacement. Notably, we observed a decrease in slit pore width although the expression of slit pore proteins was unchanged. Also, the acute albuminuria could not be related to alterations in cytoskeleton-associated proteins, the GBM adhesion molecule alpha(3)-integrin or heparan sulfate in the GBM. The dose-dependent development of FGS and the correlation between the percentage FGS and time-averaged albuminuria suggest that, in our model, FGS is a consequence of podocyte injury. However, the data leave open the possibility that albuminuria itself contributes to FGS development. The Thy-1.1 transgenic mouse model is an excellent model to study further the relationship between podocytic injury, albuminuria and the development of FGS.