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.     

Lung tumorigenicity in A/J and rasH2 transgenic mice following mainstream tobacco smoke inhalation.

Hypothesizing that their respective genetic backgrounds would confer an increased sensitivity to lung tumorigenesis, the plausibility of selected rodent models for the inhalation testing of mainstream tobacco smoke (MTS) was evaluated. Strain A/J and rasH2 transgenic (Tg) mice were exposed to MTS from Kentucky 1R4F research cigarettes using either a whole-body or nose-only exposure regimen. The whole-body regimen consisted of a 20-week exposure period [0.200 mg wet total particulate matter/liter (WTPM/l), 6 h/day, 5 days/week]; nose-only dosing proceeded for 28 weeks [0.040, 0.125, or 0.400 mg WTPM/l, 3 h/day, 5 days/week]. Both regimens included a 16-week recovery period. Gross and microscopic examinations of the lungs were used to evaluate tumor formation, with experimental results supporting the following conclusions: 1. Evaluation of MTS-induced tumorigenicity based on gross evaluation versus microscopic confirmation provides strikingly disparate results, indicating that serial sectioning is necessary for a definitive assessment of lung tumors. 2. While the dosing regimens employed do not allow for a definitive comparison, whole-body exposure appeared to be more effective for inducing statistical changes in tumor multiplicity and incidence compared to nose-only exposure. 3. Exposure-related stress, evidenced as reductions in both body weight gain and background tumor formation, represents a potential confounder during inhalation testing of MTS tumorigenicity, with additional investigation warranted to validate the specificity of exposure-related responses. 4. Comparative findings between A/J and rasH2 Tg mice suggest that the former may be overly sensitive to exposure-related stress, potentially influencing tumorigenic responses.     

转基因鼠晶状体特异性表达的制瘤素对眼发育的影响

目的 研究晶状体特异性表达的制瘤素（oncostatin M ，OSM )对眼发育的影响。 方法 将去除部分序列的小鼠OSM cDNA［661碱基对（base pair,bp)］ 连接到αA-晶状体蛋白 （αA-crystallin）启动子上，然后用显微注射的方法将其导入单细胞胚胎内，建立转基因鼠。常规组织学及免疫组织化学方法对转基因鼠进行特性鉴定，末端脱氧核苷酸转移酶介导的脱氧尿嘧啶末端标记（terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling,TUNEL ）试验检测细胞凋亡，原位杂交检测caspase-3-mRNA的表达，兔抗活化的caspase-3 抗体检测活化的caspase-3蛋白质。 结果 胚胎期14.5、17.5 d，转基因蛋白OSM特异性表达于晶状体纤维细胞内，从胚胎期17.5 d开始，转基因鼠视网膜开始发生变性，出生时，50％以上的视网膜细胞丢失。TUNEL试验显示转基因鼠视网膜细胞凋亡。转基因鼠视网膜细胞中caspase-3被激活。 结论 晶状体特异性的OSM表达激活caspase-3，导致了眼的异常发育、细胞凋亡及广泛的视网膜变性。 （中华眼底病杂志，2003，19：201-268）     

新制抗癌方合剂对染毒AFB1的HBV转基因小鼠肝癌变指标的影响

目的研究新制抗癌方合剂对染毒AFB,的HBV转基因小鼠肝癌变的预防作用及对氧自由基代谢的影响.方法运用透射电镜等形态学观察方法和生化检测法研究新制抗癌方合剂对染毒AFB1的HBV转基因小鼠肝癌变的预防作用和对氧自由基代谢的影响.结果模型对照组小鼠肝脏结节发生率和结节数目显著高于中药预防组(均P＜0.01),模型对照组小鼠肝小叶结构消失,被大量不典型增生灶及增生结节取代,出现了大量异形细胞,异形细胞形态基本接近癌细胞,中药预防组小鼠肝小叶结构存在,可见少量肝细胞不典型增生灶和增生结节,异形细胞显著减少.中药预防组小鼠肝组织SOD、GST酶活性高于模型对照组,而MDA含量低于模型对照组(P＜0.05或P＜0.01).结论新制抗癌方合剂具有阻断AFB1协同HBV致肝癌变作用,其机理与该方提高肝组织SOD和GST酶活性,降低MDA含量有关.     

上调星形胶质细胞中PP2A对APP/PS1双转基因小鼠的神经保护作用

目的:探讨上调星形胶质细胞中蛋白磷酸酶2A(protein phosphatase 2A,PP2A)对APP/PS1双转基因小鼠的神经保护作用。方法:构建带胶质细胞原纤维酸性蛋白启动子的e GFP-wt PP2A慢病毒,特异性上调星形胶质细胞中的PP2A。将实验小鼠随机分为野生对照+慢病毒空载体组(Con组)、APP/PS1双转基因小鼠+慢病毒空载体组(APP/PS1组)和APP/PS1双转基因小鼠+慢病毒感染组(PP2A组),各组小鼠经侧脑室注射慢病毒4周后,采用脑片免疫荧光检测β-淀粉样蛋白(β-amyloid protein,Aβ)的水平,通过Golgi染色观察树突棘密度和形态学变化,电镜检测突触后致密物(postsynaptic density,PSD)的厚度,Morris水迷宫定位航行实验检测感染慢病毒后对学习和记忆的影响。结果:上调星形胶质细胞中PP2A降低APP/PS1双转基因小鼠Aβ的水平,增加树突棘密度、有突触功能的蘑菇状树突棘比例和PSD厚度,缩短寻找平台逃避潜伏期。结论:上调星形胶质细胞中PP2A改善APP/PS1双转基因小鼠AD样Aβ聚集的病理改变,具有重塑突触结构与功能和改善学习记忆能力的作用。