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     

用显微注射法建立转bcl—x1基因小鼠

建立转bcl-x1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-x1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1654枚，移植卵数1448枚，受体鼠49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0．24％。初步获得了转bcl-x1基因小鼠。     

转突变型淀粉样蛋白前体双荧光融合基因阿尔茨海默病小鼠模型的建立及初步鉴定

目的 探索建立转突变型淀粉样蛋白前体(APP)双荧光融合基因(CFP-54 bp-YFP-C99)转基因小鼠模型的可行性和基因整合效率.方法 采用显微注射法,将构建的突变型APP双荧光融合基因注入昆明小白鼠的受精卵,获取子代鼠,通过聚合酶链反应(PCR)方法初步鉴定基因的整合情况,以获得阳性鼠.结果 注射昆明小白鼠受精卵共2202枚,移植1806枚.受体鼠56只,怀孕鼠13只,共生产52只子代鼠,其中存活32只.PCR初步检查整合有突变型APP双荧光融合基因的阳性鼠2只,均为活鼠.移植鼠的总怀孕率为23.2%(13/56),突变型APP双荧光融合基因的整合效率为3.9%(2/52).结论 显微注射法建立转突变型APP双荧光融合基因的阿尔茨海默病小鼠模型是可行的,但转基因效率较低.     

Mdx小鼠骨骼肌纤维化及Spp1基因表达

目的通过观察Duchenne型肌营养不良的模型鼠mdx小鼠骨骼肌中纤维化情况,研究Spp1基因在mdx小鼠及其对照鼠中不同时期的表达,探讨在mdx小鼠中Spp1与肌纤维化的关系。方法选取雄性C57BL/10Sc Sn-Dmdmdx/JNju鼠为实验组,雄性C57BL/6Sc Sn小鼠为对照组,根据年龄分为2w组、4w组、8w组、12w组。每组选取6只,3只用于冰冻切片的苏木精-伊红染色及马松(masson)染色,3只用于基因芯片及q RT-PCR。结果 2w及4w时mdx小鼠无明显结缔组织增生,8w时,mdx小鼠可见轻度结缔组织增生;12w时,mdx小鼠结缔组织增生程度较8w稍加重,仍为小片状区域的纤维化,对照组小鼠不同时期均未见纤维化;mdx小鼠2w与12w股四头肌基因芯片表达谱对比,其中Spp1基因在mdx小鼠2w与12w相比fold-change值为-15.1354,变化明显;Spp1在mdx小鼠股四头肌不同时期表达量比较:8w组较4w组明显升高,12w组较8w组表达量下降,但仍高于4w组,8w组与12w组Spp1表达量较同期对照组明显升高,差异具有统计学意义。结论 mdx小鼠早期(2w~4w)肌纤维化表现不明显,8w时骨骼肌内可见少量结缔组织增生,随后缓慢进展。2 Spp1基因在mdx小鼠成熟期(8w~12w)表达量明显增加,推测其在mdx小鼠肌纤维化中发挥一定作用。     

胰岛素样生长因子1基因转染成肌细胞促小鼠胫骨骨折愈合的作用

背景：随着基因治疗的研究发展,将转有胰岛素样生长因子1基因的细胞移植入损伤区,达到该基因在体内的持续表达已可行,并有可能解决骨折患者骨不连及骨延迟愈合等难题。 目的：观察携带人胰岛素样生长因子1基因的成肌细胞体内局部多点注射对C57BL/6J小鼠胫骨骨折愈合的影响。 设计、时间及地点：随机对照动物实验,于2007-01/2008-04在汕头大学医学院完成。 材料：8周龄雄性C57BL/6J小鼠42只,体质量22~25 g,建立左胫骨骨折的动物模型。 方法：42只小鼠随机数字表法分为2组,每组21只。实验组于骨折处周围肌肉中多点注射0.3 mL转人胰岛素样生长因子1基因成肌细胞悬液;对照组注射0.3 mL生理盐水。分别在造模后2,3,4周每组各随机取7只小鼠处死,取材,行苏木精-伊红染色。 主要观察指标：各组动物骨折部位形成骨痂面积的大小和组成成分的变化;外源性细胞在体内存活及表达人胰岛素样生长因子1情况;各组动物骨密度及骨痂细胞生长情况。 结果：①实验组转基因成肌细胞BrdU及人胰岛素样生长因子1免疫组织化学染色均阳性,4周实验组平均灰度值虽稍高于2周实验组,但差异无显著性意义(P > 0.05)。②携人胰岛素样生长因子1基因的成肌细胞移植入C57BL/6J小鼠胫骨骨折处周围肌肉中可存活4周以上,并能稳定地分泌人胰岛素样生长因子1。③实验组造模后各时间点外骨痂中小梁骨占骨痂总面积的百分比均高于对照组,其中第3,4周经F检验差异有显著性意义(P < 0.05);4周实验组骨痂骨密度高于对照组(P < 0.05)。④电镜观察可见实验组骨生成细胞的数量、活跃程度和胶原纤维的数量、分布均优于对照组。 结论：局部注射转人胰岛素样生长因子1基因的成肌细胞可以促进C57BL/6J小鼠胫骨骨折的愈合。     

SOD1-G93A小鼠中枢神经系统SIRT1的变化及白藜芦醇对其的影响

目的观察SOD1-G93A小鼠运动皮质和腰髓中SIRT1的变化以及白藜芦醇对SIRT1的影响。方法利用SOD1-G93A小鼠及其野生型小鼠作为实验对象,分别检测随着疾病进展小鼠神经系统中SIRT1表达的动态变化及给予白藜芦醇后SIRT1的变化。结果随着ALS疾病进展,症状早期及终末期SIRT1表达增多,而给予白藜芦醇后,SIRT1表达无明显变化,给予溶剂后SIRT1表达增多。结论随着疾病进展,SOD1-G9A小鼠运动皮质与腰髓中SIRT1的表达逐渐增多;白藜芦醇对SIRT1未起到激活作用,白藜芦醇在SOD1-G93A小鼠模型中有无有益作用尚不能定论。     

应用基因编辑技术探索建立中枢神经系统动静脉畸形小鼠模型

目的采用CRISPR/Cas9技术编辑小鼠Alk1基因,探讨条件性敲除Alk1建立中枢神经系统动静脉畸形动物模型的可行性。方法利用CRISPR/Cas9技术在小鼠Alk1基因的特定位点插入两个Lox P序列;经传代并与SM22-Cre小鼠交配后培育出Alk1~(2Lox P/2Lox P)/Cre目标小鼠。该目标小鼠8周龄时通过他莫昔芬腹腔注射激活Cre重组酶的表达,从而条件性敲除Alk1基因诱导脑动静脉畸形的生成。2周后取小鼠脑组织标本,进行苏木精-伊红染色和病理分析。结果 7只目标小鼠中,2只脑组织病理切片发现管腔大小不一的异常血管病灶。结论条件性敲除Alk1基因可初步建立中枢神经系统动静脉畸形动物模型。     

转基因小鼠中bcl-xl基因的过表达在小鼠大脑中动脉栓塞中的保护作用

目的观察bcl-xl基因的过表达对小鼠大脑中动脉栓塞的保护作用,探讨其作用机制。方法通过建立bclxl转基因小鼠,经传代及检测后证实,该转基因小鼠中存在着bclxl基因的过表达,然后将该模型小鼠与同种系野生型小鼠同时行线栓永久性阻塞大脑中动脉,在缺血24h时测其神经功能评分,观察转基因小鼠与野生型小鼠的差别。在缺血后不同时间点测其梗死体积,观察梗死体积的动态变化。用TUNEL法观察小鼠的脑组织缺血后不同时间点再灌注时凋亡细胞的数量和分布情况。用免疫组化方法观察梗死前后两种小鼠脑组织中bcl-xl的表达量的差异。结果缺血24h后转基因小鼠的神经功能评分低于野生型小鼠(P<0.05)。缺血后3、24、72h,转基因小鼠的梗死体积均明显低于野生型小鼠,差异有显著性意义。TUNEL显示在缺血再灌注后的不同时间点,转基因小鼠皮质缺血区内的凋亡细胞数明显少于野生型小鼠,差异有显著性意义。免疫组化结果显示,梗死前后转基因小鼠的皮质细胞bclxl的表达量均明显高于野生型小鼠(P<0.05),且梗死后两种小鼠体内的bclxl的表达量均较梗死前增加(P<0.05)。结论在规范化的标准条件下,转基因小鼠中bclxl基因的过表达能够降低脑梗死的体积并改善小鼠的神经功能;过表达bclxl基因的这种效应可能是通过抑制细胞凋亡而实现的。     

转VEGF基因内皮祖细胞移植治疗阿尔茨海默病模型小鼠的研究

目的 将体外诱导的血管内皮祖细胞（EPCs）转入血管内皮生长因子（VEGF）基因，并定 向移植到AD 模型鼠脑内海马区，观察该部位及邻近部位的血管重建以及内皮细胞功能的改善情况，为 AD的治疗提供研究基础。方法 采用体外分离培养小鼠骨髓源性EPCs。构建的携带人VEGF165 基 因的pcDNA3.1 质粒转染至EPCs，观察EPCs 表达VEGF 情况。采用APP/PS1 双转基因鼠模型，将转染人 VEGF165 基因的EPCs 移植至模型鼠脑内，观察小鼠行为学的变化、Aβ 含量，以及血管的形成情况。结 果 将转染VEGF基因的EPCs移植入小鼠的海马区后，动物在第4，5，6 天进行空间导航试验发现，与 对照组比较，平均潜伏期明显缩短（P＜ 0.05）；免疫组化结果显示，与对照组、EPCs 组比较，EPCs+VEGF 组额叶皮层和海马区Aβ含量明显减少（P＜ 0.01），海马区CD34 阳性细胞数更高（P ＜ 0.01）。结论 将 EPCs+VEGF 移植入鼠脑内，可以发现小鼠的记忆能力增强，原因可能与促进新生血管的生成、Aβ 清除 有关。     

强力霉素调控小鼠E1A激活基因阻遏子表达NIH3T3细胞系的建立***☆

背景：前期研究发现，E1A激活基因阻遏子蛋白调控人血管平滑肌细胞的增殖及表型转化，但其表达调控细胞增殖、分化、凋亡的量效关系有待深入探讨。 目的：建立强力霉素调控小鼠E1A激活基因阻遏子表达的NIH3T3细胞系，拟为定量观察E1A激活基因阻遏子的生物学功能提供研究基础。 设计、时间及地点：对比观察实验，于2006-05/2008-03在沈阳军区总医院全军心血管病研究所实验室完成。 材料：强力霉素调控基因表达系统，3T3细胞系。 方法：通过反转录-聚合酶链反应方法获得小鼠E1A激活基因阻遏子（mCREG） cDNA序列；将mCREG cDNA序列亚克隆入pRev-TRE载体中，构建强力霉素调控E1A激活基因阻遏子表达pRev-TRE-mCREG载体；分别经G418（新霉素）及潮霉素筛选表达pRev-Tet-On、pRev-TRE-mCREG的NIH3T3细胞克隆；反转录-聚合酶链反应鉴定转染细胞克隆中抗性基因和插入基因的表达。 主要观察指标：Western blotting检测不同浓度的强力霉素诱导后NIH3T3细胞中mCREG的表达。 结果：成功构建了强力霉素可调控表达的pRev-TRE-mCREG重组载体；筛选出稳定表达双抗性基因的NIH3T3-mCREG细胞克隆；反转录-聚合酶链反应检测证实细胞克隆中G418及插入基因表达均为阳性；Western blotting检测发现随强力霉素诱导剂量的增加(0，0.01，0.1，1 mg/L)，NIH3T3-mCREG细胞中mCREG表达呈剂量依赖性增加。 结论：成功建立强力霉素调控E1A激活基因阻遏子表达的小鼠NIH3T3细胞系。     

Stereotaxic microinjection of HSV-1 selectively decreases striatal dopamine concentrations in mice

BALB/c mice were stereotaxically injected in the striatum with either the MP or MacIntyre strain of herpes simplex type 1. Three days later, at a time when the animals were free from overt signs of infection, they were killed by cervical dislocation and the striatum was rapidly removed. Concentrations of dopamine, norepinephrine, serotonin and their metabolites were subsequently determined by means of high-performance liquid chromatography with electrochemical detection. With both strains of virus, dopamine levels were reduced and the ratio of homovanillic acid to dopamine was elevated in MP-inoculated mice. Norepinephrine, serotonin and its metabolites were unaffected. Immunoperoxidase staining in separate, identically treated animals indicated that the infection was confined to the striatum and necrosis was minimal at this point in time. These results demonstrate that dopamine metabolism can be affected by herpes simplex in the absence of immunocytochemical evidence of infection of the cell bodies of dopaminergic neurons or cellular death.     

Functional phenotype in transgenic mice expressing mutant human presenilin-1

Mutations in the presenilin-1 (PS1) gene cause approximately 50% of cases of early onset familial Alzheimer's disease. The function of this protein remains unknown. We have made an electrophysiological study of hippocampal slices from transgenic mice expressing either a normal human PS1 transgene (WT) or one of two human PS1 transgenes bearing pathogenic mutations at codon M146 (M146L and M146V). Medium and late afterhyperpolarizations in CA3 pyramidal cells were larger in mice expressing either mutant form compared with WT and nontransgenic controls. Calcium responses to depolarization were larger in M146L mice compared with nontransgenic littermates; synaptic potentiation of the CA3 to CA1 projection was also stronger. These results demonstrate disruption of the control of intracellular calcium and electrophysiological dysfunction in PS1 mutant mice.     

Paradoxical locomotor behavior of dopamine D1 receptor transgenic mice

The behavioral effects of augmenting dopamine D1 receptor expression in the brain were investigated in mice incorporating additional copies of the mouse D1 receptor gene. Two transgenic lines showed increases in brain D1 receptor binding sites, which were greatest in extrastriatal regions. The full D1 agonist SKF 81297, when administered systemically to control animals, stimulated a dose-dependent increase in locomotor activity. In contrast, in D1 receptor overexpressing transgenic mice, this drug caused a marked suppression of locomotion due to a decrease in the frequency of movement initiation. Amphetamine and cocaine induced comparable locomotor activation in both transgenic animals and their control littermates. In the transgenic animals, D1 agonist-induced rearing and climbing behaviors were suppressed. However, on rotarod testing, the agonist-treated transgenic and control mice performed comparably, indicating that sensorimotor coordination was unaffected. These studies demonstrate that altering the levels of D1 receptor expression reverses the effects of D1 agonism on locomotor initiation and rearing.     

Polysomnography in transgenic hSOD1 mice as Down syndrome model

Sleep-wake homeostasis is crucial for behavioral performances and memory in the general population and in learning disability populations among them Down syndrome patients. We investigated, in a mouse model of Down syndrome, cortical EEG and sleep-wake architecture under baseline conditions and after a 4 hr sleep deprivation (SD). Young heterozygous transgenic mice (S/+) for the human Cu/Zn superoxide dismutase (hSOD-1) were obtained on FVB/N background. Baseline records for slow wave sleep (SWS) and wake (W) parameters were the same in S/+ and control mice whereas paradoxical sleep (PS) episode number decreased and PS latency increased after light off in S/+ mice. These data correlate well the polysomnographic phenotype of young DS patients.     

Galanin overexpressing transgenic mice

Galanin overexpressing transgenic mice (GAL-tg) were generated on two different promoters. Both lines of GAL-tg displayed high levels of galanin in the hippocampus and reduced sensitivity to seizures, as compared to their respective wildtype littermate controls (WT). Performance deficits on learning and memory tasks, impaired long-term potentiation, reduced hippocampal excitability, lower evoked glutamate release, and reduced numbers of choline acetyltransferase immunoreactive neurons in the horizontal limb of the diagonal band were detected in GAL-tg as compared to WT. Changes in sensitivity to nociceptive stimuli were demonstrated in one line. GAL-tg represent a new model for investigating the biological actions of endogenous galanin, and for testing novel therapeutics based on galanin receptor ligands.     

Impaired spatial navigation learning in transgenic mice over-expressing heme oxygenase-1

Transgenic mice expressing heme oxygenase-1 (HO-1) using the neuron-specific enolase promoter were impaired in learning the Morris water maze compared to nontransgenic littermates. The memory of the HO-1 mice for the location of the platform was similarly impaired when tested using a probe trial after 7 training blocks, but performance on visible platform trials was similar for both groups of mice. Importantly, both HO-1 and nontransgenic mice had normal sensorimotor function, and performed the same on a Y-maze alternation task, highlighting the specificity of memory deficit in the spatial navigation task. These results suggest that carbon monoxide, one product of HO-1 activity, interferes in the development of spatial navigation memory, and may play a role in normal memory function.     

SOD1-G93A转基因小鼠的鉴定方法比较

目的建立一种简便、节省和准确的肌萎缩侧索硬化(ALS)hSOD1-G93A转基因小鼠DNA提取及基因鉴定方法。方法以B6SJL-BTg(SOD1-G93A)1Gur/J半合子雄鼠与B6SJLF1雌鼠交配产仔,剪尾尖约1mm,提取组织DNA,PCR扩增hSOD1基因片断,电泳后观察结果并对PCR产物进行纯化、测序及BLAST验证。同时留取鼠尾血50～100μl,提取血DNA并PCR扩增作为对照,比较两者之间的符合率。结果hSOD1-G93A阳性鼠约占54%,基本符合孟德尔遗传规律。鼠尾组织及血DNA鉴定结果符合率为100%。结论鼠尾尖组织DNA提取及鉴定是一种简便易行、节省经费并且准确可靠的方法。     

Reduced anxiety-related behaviour in transgenic mice overexpressing serotonin 1A receptors

Serotonergic neurons play a major role in the modulation of emotion and behaviour. Especially knockout studies have revealed a role for the serotonin(1A) (5-HT(1A)) receptor in anxiety related behaviour. Mutant animals exhibit enhanced anxiety-like responses, possibly resulting from impaired autoinhibitory control of midbrain serotonergic neurons. To further elucidate the role of the 5-HT(1A) receptors in affective behaviour, a complementary approach has been used and transgenic mice overexpressing this receptor subtype have been generated. The expression of the active 5-HT(1A) receptor protein as indicated by autoradiography was transiently increased during early postnatal development (P1.5) as compared to wild-type mice. Within the next 2 weeks, the increase in receptor binding vanished and was also not apparent in adult animals indicating adaptive changes in the regulation of 5-HT(1A) receptor expression. Although no evidence for increased receptor binding in the brains of adult homozygous mice was found by autoradiography, typical phenotypic changes indicative of 5-HT(1A) receptor overactivity were apparent. Transgenic mice revealed a reduced molar ratio of 5-hydroxyindoleacetic acid to serotonin in several brain areas and elevated serotonin values in the hippocampus and striatum. Moreover, anxiety-like behaviour was decreased in male and female transgenic mice and body temperature was lowered in male transgenic mice in comparison with heterozygous and wild-type mice. These findings further underline the pivotal role of 5-HT(1A) receptors in the homeostasis of anxiety-like behaviour and the crucial importance of stimulation of the 5-HT(1A) receptor during the early postnatal development for normal anxiety-like behaviour throughout life.