神经组织特异表达绿色荧光蛋白转基因小鼠的脑组织形态观察

目的 建立神经组织特异表达绿色荧光蛋白的转基因小鼠,为神经系统的形态学观察提供可以荧光示踪的工具动物.方法 把增强型绿色荧光蛋白(EGFP) 因插入血小板源性生长因(PDGF)B-链启动子下游构建转基因载体,用显微注射的方法建立转基因C57BL/6J小鼠.PCR鉴定转基因小鼠的基因表型,对阳性转基因小鼠的脑组织进行矢状面冷冻切片,分别进行HE染色,显微镜观察组织结构,荧光体视镜及荧光显微镜观察绿色荧光蛋白(GFP)在神经组织的表达.结果 在8个首建品系中筛选出1个神经组织高表达绿色荧光蛋白的转基因小鼠系.观察到绿色荧光蛋白在大脑皮层、海马、丘脑、小脑及脑干等部位表达.结论 建立了稳定遗传的神经组织特异表达绿色荧光蛋白转基因小鼠品系,为神经系统的生理学及病理学研究提供了可以荧光示踪的模型动物.     

一种新型荧光(mKate2)特异性标记中性粒细胞的转基因小鼠的构建及初步应用

目的构建在中性粒细胞中特异性表达mKate2红色荧光蛋白的转基因小鼠。方法本研究将人工合成的lysozyme M启动子及mKate2基因序列构建至pmini Tol2系统中,通过显微注射法把线性化的转基因载体注射到C57BL/6J小鼠的受精卵后并将其移植至假孕鼠体内,制备转基因小鼠。本研究使用PCR鉴定lyz-m Kate2转基因小鼠的成功构建和通过荧光显微镜和流式分析等方法鉴定该模型小鼠中荧光蛋白mKate2的表达及特异性。结果外源lyz-mKate2转基因盒在C57BL/6J小鼠体内成功表达。在外周血中检测到标记红色荧光信号的中性粒细胞,而且70%左右的mKate2阳性细胞是中性粒细胞。激光诱导血栓模型中血栓块可观察到mKate2阳性细胞。结论本研究成功构建了mKate2特异性标记中性粒细胞的新型转基因小鼠并验证了其在血栓模型中的应用价值。     

四环素调控的小鼠LAIR-1/CD305转基因小鼠的初步建立

目的：建立四环素调控的小鼠LAIR-1/CD305转基因小鼠,为进一步研究mLAIR-1分子的体内功能奠定基础.方法：构建pBI-5-mLAIR-1载体,显微注入B6D1F1受精卵,PCR检测新生小鼠基因组DNA中LAIR-1与荧光素酶（Luciferase）基因.将mLAIR-1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含盐酸强力霉素（Dox）的培养基进行培养,检测细胞裂解液中荧光素酶活性.将荧光素酶表达依赖Dox小鼠与C57BL/6交配,采用PCR对子代鼠进行检测.结果：共获得9只首建鼠,其目的基因表达高度依赖Dox,并得到其中5只首建鼠的F1代小鼠.结论：获得了四环素调控的小鼠LAIR-1转基因小鼠,可用于该分子体内功能的研究.     

实时荧光定量PCR检测转基因小鼠外源基因拷贝数方法的建立和应用

目的建立实时荧光定量PCR方法检测CaMKⅡα-Cre转基因小鼠外源基因拷贝数的方法。方法以CaMKⅡα-Cre转基因首建鼠及其仔代阳性鼠为研究对象,利用绝对定量的实时荧光定量PCR法检测转基因小鼠的拷贝数,并筛选出纯合子小鼠再经遗传育种方法以确定为纯合子小鼠。结果绝对定量标准曲线公式为:△Ct=-2.402log5N(拷贝数)+8.654,相关系数为0.9999,扩增效率为95.4%。三只CaMKⅡα-Cre首建鼠的拷贝数分别为19、7、5;三个转基因小鼠品系均成功获得纯合子小鼠。结论成功建立了检测转基因小鼠外源基因拷贝数的实时荧光定量PCR方法,该方法可用于检测各种转基因小鼠中外源基因的拷贝数。     

四环素调控的PTA1/CD226转基因小鼠的初步建立

为研究小鼠PTA1分子在体内的功能,建立四环素调控的小鼠PTA1/CD226转基因小鼠,我们构建了pBI-5-mPTA1载体,显微注射入B6D1F1受精卵,使用PCR检测新生小鼠基因组DNA中的PTA1与荧光素酶(luciferase)基因。将mPTA1和荧光素酶双阳性小鼠耳成纤维细胞转染含rtTA的pUHD17.1质粒,用含有盐酸强力霉素(Dox)的培养基进行培养,检测细胞裂解液中荧光素酶的活性。将荧光素酶表达依赖Dox的小鼠与C57BL/6小鼠交配,采用PCR对子代鼠进行检测。最终共获得7只首建鼠,其目的基因表达高度依赖Dox,并得到了其中2只首建鼠的F1代小鼠。     

PD-L1转基因小鼠的建立及其脊髓损伤后的运动功能恢复

目的:建立广泛表达PD-L1的转基因小鼠,并且评价转基因小鼠脊髓损伤后的运动功能恢复情况。方法:克隆小鼠PD-L1基因编码区的cDNA,经测序正确后,将PD-L1基因cDNA插入到pCAGGS质粒中,构建pCAG-PD-L1转基因载体;经C57BL/6小鼠受精卵原核注射,建立PD-L1转基因小鼠;PCR鉴定转基因小鼠的基因型;以流式细胞术(FCM)检测转基因小鼠脾脏中T、B淋巴细胞PD-L1的表达情况;免疫组织化学检测转基因小鼠周围组织PD-L1表达情况;RT-PCR检测转基因小鼠神经组织内PD-L1表达;BBB评价转基因小鼠脊髓损伤后运动功能恢复。结果:获得PD-L1转基因小鼠,外源PD-L1基因可以顺利遗传给子代;免疫组织化学、RT-PCR和FCM发现:PD-L1转基因小鼠的神经组织、淋巴组织以及生殖器官中高表达PD-L1;PD-L1转基因小鼠脊髓重度夹伤后,其BBB运动学评分明显高于野生型小鼠(P<0.05)。结论:成功地建立了C57BL/6背景的PD-L1转基因小鼠,且PD-L1基因的高表达促进脊髓损伤后的运动功能恢复。     

LMO4在阿尔茨海默氏病转基因小鼠海马的表达

目的检测LMO4在APP/PS1-Tg转基因小鼠脑组织中的表达。方法选取6月龄大小的APP/SP1-Tg转基因小鼠为实验组,同月龄同种系的野生型小鼠C57BL/6为对照组,分别提取其脑组织海马mRNA和蛋白,应用实时荧光定量PCR和蛋白免疫印迹方法检测两组小鼠脑组织中LMO4蛋白和基因的表达。结果 LMO4蛋白和基因在APP/SP1-Tg转基因小鼠海马组织表达水平明显低于月同种系的野生型C57BL/6小鼠海马组织表达水平(P<0.05)。结论 LMO4在APP/SP1-Tg转基因小鼠低表达可能与阿尔茨海默氏病的发病过程相关。     

活体动物光学成像技术与应用研究

活体动物光学成像是利用生物发光及荧光技术在活体动物体内进行生物标记通过光学成像系统来监测被标记动物体内分子及细胞等的生物学过程。按发光模式可分为生物发光和荧光两类。相对于传统动物实验研究方法,具有无创、可多次重复、实时活体成像、灵敏、安全等优势,这项技术在标记活体内肿瘤活体细胞示踪、标记基因及转基因动物等方面的应用广泛。     

小鼠精原细胞移植技术的改进

目的 对现有小鼠精原细胞移植技术进行改进,建立更具操作性,易于推广的新方法。方法 分离含绿色荧光蛋白(GFP)的rC57BL6/tg14 (act-EGFO-Osb Y01)小鼠精原细胞作为供体细胞,应用自制的显微注射器将其从睾丸的精子输出管处,以锥虫蓝作为指示剂直接注入野生型预处理C57BL6小鼠精细管内完成移植。应用荧光显微镜观察移植效果并进行组织学分析。结果 移植后受体小鼠睾丸出现绿色荧光信号,组织学检查发现精细管中荧光信号显著高于周围组织,移植的供体小鼠（GFP）的睾丸细胞在受体小鼠睾丸中形成克隆和形成精子,而预处理的野生型小鼠睾丸精细管未发现。结论 含GFP蛋白的rC57BL6/tg14小鼠精原细胞在受体小鼠体内移植成功,此改进方法在简化精原细胞移植技术的同时可保证移植质量。     

SV40T胃壁细胞定位表达转基因小鼠的建立

目的 构建胃壁细胞定位表达SV40T的真核表达载体并制备转基因小鼠动物模型,为研究胃癌发病机制提供动物模型.方法 从构建的胃壁细胞特异性表达载体pcDNA3.1(-)/HKSV中酶切回收3.8kb的基因片段H -K ATPase β promoter/SV40T,通过显微注射的方法制备转基因小鼠,PCR和Southern blotting检测阳性转基因小鼠并建系繁殖,RT-PCR检测基因的表达情况.结果 将422枚注射过的受精卵移植给16只假孕雌鼠,共生出77只仔鼠,移植成功率为18.2%.在出生的77只仔鼠中,2#、4#、8#、16#、24#、51#、57#、61#、68#、73#经PCR检测为阳性首建鼠.除68#不育外,其他9个品系首建鼠共生出99只F1代鼠.8#品系23只F1代尚未发现阳性鼠,另8个品系F1代经PCR和Southern blotting检测发现31只阳性鼠,阳性率为40.8%(31/76).RT-PCR检测F1代阳性鼠均仅在胃组织中有SV40T基因的表达,而在心、肝、肾、肺、食道、肠、骨骼肌等组织中均不表达.不育首建鼠处死解剖发现胃组织有肿瘤存在.结论 建立了胃壁细胞定位表达SV40T基因的转基因小鼠动物模型.     

HBD-2转基因小鼠的建立及鉴定

人β防御素2（Human beta defensin 2，HBD-2）是人体抗菌肽的重要分子，体外试验研究证明具有广谱抗微生物活性，为开展其在整体水平上的功能研究，建立HBD-2转基因小鼠模型。用分子克隆方法构建了带CMV启动子的HBD-2全长cDNA真核重组表达质粒pCDNA3．1-HBD2，以此为摸板，PCR扩增出带CMV启动子和BGH polyA尾的HBD-2基因片段，用显微注射技术将此微基因导入小鼠雄原核中，于M16培养液培养后经输卵管移植到受体鼠体内让其怀孕产生子代小鼠。PCR法检测外源基因在小鼠基因组中的整合，RT—PCR和免疫组化法检测HBD-2在小鼠组织的表达。PCR检测结果显示17只F1代转基因鼠中4只检测到阳性信号，RT—PCR和免疫组化检测显示HBD-2在其F1代转基因小鼠生殖道、呼吸道、泌尿道以及血管内皮等组织部位广泛表达。实验表明HBIN2基因已整合到小鼠基因组且广泛表达，为进一步研究HBD-2的生物学功能及基因调控提供了有用的整体动物模型。     

HLA—Ⅱ类基因DRα,DRB1＊0401转基因鼠模型的建立及体内表 …

OBJECTIVE: To construct transgenic mice model on DR alpha and DRB1*0401 of human MHC-II molecules. METHODS: By microinjection techniques on germ nucleus of zygotes, DRalpha and DRB1*0401 genes were injected into zygotes of C57BL/6 x DBA/1 hybrid mice and transplanted into the oviducts of pseudopregnancy female mice. The integration and expression of exogenous genes in offspring were detected by PCR, Southern blot and Northern blot, RT-PCR analysis. RESULTS: There were 5 founders of all injected mice, which had steadily inherited to the fifth generation. It was found that 95 mice were positive by PCR and 68 mice were integrated exogenous gene by Southern blot analysis. DRalpha and DRB1*0401 genes were expressed on spleen and kidney of transgenic mice. CONCLUSION: This experiment on the construction of DRalpha and DRB1*0401 transgenic mice model is a success.     

APPSWE转基因鼠的繁育及子代鉴定的研究

目的:探讨老年性痴呆(AD)转基因模型鼠(TgAPPSWE2576杂合子鼠,Tg2576)的优化繁育方法,鉴定APPSWE基因阳性的子代鼠,为下一步AD研究奠定基础。方法:(1)用3种不同的交配方式观察子代鼠的存活率及APPSWE基因阳性的比率;(2)从子鼠鼠尾中提取基因组DNA,用PCR方法扩增APPSWE基因片段,电泳后观察结果;(3)PCR产物装入pGEM-T easy载体中,测序证实其基因序列。结果:雄性和雌性Tg2576互交,所产4窝子鼠,2 d内全部死亡。雄性Tg2576和雌性C57BL交配所产子鼠存活率81%,APPSWE基因的阳性率为43.3%。雌性Tg2576和雄性C57BL交配存活率82.4%,APPSWE基因的阳性率21.9%。经秩和检验两种繁育方式所产子鼠的存活率无显著差异(P＞0.05),APPSWE基因的阳性率有显著差异(P＜0.05)。琼脂糖电泳显示PCR产物分子量为428 bp,与目的基因片段相对分子质量大小一致;DNA序列测定证实PCR产物基因序列和APP片段基因序列一致,并存在APPSWE(K670N,M671L)突变。结论:雄性Tg2576和雌性C57BL品系小鼠交配是繁育APPSWE基因阳性子鼠的较好方法;本实验所用PCR方法能够精确鉴定APPSWE基因阳性子鼠,为AD的实验研究提供了较理想的动物模型。     

四种细胞移植重建C57BL雌鼠免疫功能的对比

背景：据报道肌肉来源的细胞能重建造血功能,还需要进一步观察其他系细胞是否也能重建造血功能。 目的：对比不同细胞对C57BL雌鼠造血功能恢复的作用。 方法：摘眼球取绿色荧光蛋白转基因雄鼠外周血,溶血后制成白细胞,再取脾脏、肝脏制成脾细胞和肝细胞悬液,同时制备骨髓细胞。将C57BL雌鼠半致死量射线照射后,回输4种细胞,并在移植后18,39,53 d检测外周血绿色荧光蛋白阳性细胞。同时标记CD4-PE和CD8-PE,并在移植后100 d杀鼠取骨髓检测Y染色体阳性率。 结果与结论：移植后18,39,53 d均在C57BL雌鼠外周血检测到绿色荧光蛋白阳性细胞;C57BL雌鼠外周血绿色荧光蛋白阳性细胞百分比：回输脾细胞和骨髓细胞>回输肝细胞>回输外周血白细胞。同时在回输脾细胞和骨髓细胞的C57BL雌鼠外周血中检测到绿色荧光蛋白和CD4双阳性细胞、绿色荧光蛋白和CD8双阳性细胞,而回输肝细胞和外周血白细胞的C57BL雌鼠外周血中几乎未检测到双阳性细胞。移植后100 d检测到的Y染色体阳性率与外周血中绿色荧光蛋白阳性率呈正相关。结果说明绿色荧光蛋白转基因雄鼠的外周血白细胞、脾细胞、肝细胞和骨髓细胞均有重建造血功能的作用,其中脾细胞与骨髓细胞作用相似,作用大于肝细胞,肝细胞作用大于白细胞。     

Amphetamine-induced toxicity in dopamine terminals in CD-1 and C57BL/6J mice: complex roles for oxygen-based species and temperature regulation.

In order to examine differential strain susceptibility to neurotoxic effects of amphetamine and to assess the potential role of superoxide radicals in amphetamine-induced dopaminergic damage, the drug was injected to mice with different levels of copper/zinc superoxide dismutase (Cu/Zn SOD) enzyme. Administration of amphetamine (10 mg/kg, i.p., given every 2 h, a total of four times) to wild-type CD-1 and C57BL/6J mice caused significant decreases in dopamine and 3,4-dihydroxyphenylacetic acid levels, in [(125)I]RTI-121-labeled dopamine transporters as well as a significant depletion in the concentration of dopamine transporter and vesicular monoamine transporter 2 proteins. The amphetamine-induced toxic effects were less prominent in CD-1 mice, which have much higher levels of Cu/Zn SOD activity (0.69 units/mg of protein) in their striata than C57BL/6J animals (0.007 units/mg of protein). Transgenic mice on CD-1 and C57BL/6J background, which had striatal levels of Cu/Zn SOD 2.57 and 1.67 units/mg of protein, respectively, showed significant protection against all the toxic effects of amphetamine. The attenuation of toxicity observed in transgenic mice was not caused by differences in amphetamine accumulation in wild-type and mutant animals. However, CD-1-SOD transgenic mice showed marked hypothermia to amphetamine whereas C57-SOD transgenic mice did not show a consistent thermic response to the drug.The data obtained demonstrate distinctions in the neurotoxic profile of amphetamine in CD-1 and C57BL/6J mice, which show some differences in Cu/Zn SOD activity and in their thermic responses to amphetamine administration. Thus, these observations provide evidence for possible complex interactions between thermoregulation and free radical load in the long-term neurotoxic effects of this illicit drug of abuse.     

In vitro and in vivo expression analysis of the interferon-inducible 203 gene.

The interferon (IFN)-inducible protein family 200 is encoded by structurally related genes located on mouse chromosome 1. The encoded proteins so far characterized and designated p202, p204, and pD3 contain at least one copy of a conserved 200 amino acid domain in addition to other regions that are different or missing among the various family members. We have recently characterized a cDNA clone (203 cDNA) encoding a 408 amino acid protein bearing structural similarities to p202 and p204. Here, we report its pattern of expression in vitro and in vivo. In vitro, the mRNA and protein encoded by the 203 gene were increased by IFN-alpha in several cell lines of different histologic origin. By contrast, no significant induction was observed in vivo in mice from C57BL/6 and BALB/c strains even after treatment with the IFN-inducer poly rI:rC. In addition, the constitutive expression of 203 gene was restricted to some myeloid and lymphoid tissues, namely, thymus, bone marrow, and spleen. Comparison of the expression pattern of the 203 and 202 genes in three mouse strains revealed that they exhibit a differential inducibility by IFN and a reciprocal expression pattern. The 203 mRNA was constitutively expressed in C57BL/6 and BALB/c mice and undetectable in the spleen of DBA/2 mice. The 202 mRNA was strongly induced by poly rI:rC in the spleen of DBA/2 and BALB/c mice but absent in C57BL/6 mice. Southern analysis revealed a restriction fragment length polymorphism in the 203 locus. Taken as a whole, these results demonstrate a remarkable difference in the in vivo IFN responsiveness of two members belonging to the same gene family with a similar degree of IFN inducibility in vitro. Moreover, the reciprocal expression pattern in C57BL/6 and DBA/2 mice could mean that p203 and p202 play the same role in a mouse strain in which only one of them is expressed.