DNA指纹图在小鼠ES细胞嵌合体鉴定中的应用

尝试应用多位点DNA指纹技术,检测经过胚胎干细胞(ES细胞)途径所获得的嵌合体小鼠中ES细胞在各种脏器中的嵌合情况、检测ES细胞在嵌合体小鼠中是否实现种系传递。结果表明：采用新型的人工合成的寡核苷酸多聚体探针(JL-02探针)的多位点DNA指纹图谱,具有足够的多态性和很好的稳定性。适用于鉴定ES细胞在嵌合体小鼠各种组织器官中的嵌合情况,也适用于鉴定ES细胞是否具有种系嵌合能力,尤其适用于在嵌合体研究中,最适宜的品种组合具有相同的毛色或相同的酶型。     

用大鼠心肌条件培养基建立来源于C57BL/6J小鼠的ES细胞系

报道一种新的建立C57BL/6J小鼠ES细胞系的方法。采用大鼠心肌条件培养基,在不使用饲养层细胞和白血病抑制因子(LIF)的情况下,从C57BL/6J品系小鼠中建成1个ES细胞系即MESPU 41,成系率为1.0%。MESPU 41细胞为XX型,核型正常率高达89%,表现出XX型ES细胞系少有的稳定性。进行体内分化实验时MESPU 41细胞能发生广泛分化形成畸胎瘤。嵌合体制作实验证实MESPU 41细胞具有嵌合能力,能参与胚胎的发育。采用RT-PCR方法,检测出大鼠心肌细胞有LIF mRNA的表达,这可能与其条件培养基保持ES细胞未分化状态并使X染色体稳定有关。同时,还对大鼠心肌细胞进行了永生化的尝试,共得到了4个永生化克隆,这将进一步简化ES细胞建系和培养工作,为进一步研究ES细胞在体外培养过程中的稳定性开创了新的起点。     

LIM结构域蛋白KyoT基因剔除小鼠的建立和表型分析

KyoT为一LIM结构域蛋白,可与转录因子RBP-Jk相互作用而调节其转录活性.构建了KyoT基因剔除载体,转染小鼠胚胎干细胞(ES细胞)后得到同源重组的ES细胞克隆.将此ES细胞注射入小鼠的囊胚泡得到嵌合小鼠,再经小鼠培育得到KyoT基因被剔除的小鼠.纯合子KyoT基因剔除小鼠不能表达有功能的KyoT蛋白.表型分析表明纯合子KyoT基因剔除小鼠腹腔B1B细胞数增加,提示KyoT可能参与B淋巴细胞的发育.     

小鼠囊胚的不同遗传背景对形成ES细胞集落的影响

使用正常囊胚经过内细胞团增殖后的离散程序，比较了小鼠C57BL／6品系、129品系和C57BL／6与129杂交的囊胚在形成胚胎干细胞（ES细胞）集落上的差异。C57BL／6品系的正常囊胚经培养后只有17．4％的胚胎出现ES集落，细胞生长迅速但极易分化。129品系为41．0％，细胞生长比较缓慢。而杂交鼠胚易于出现ES细胞集落，高达75．0％，有利于ES细胞系的建立。文中讨论了在嵌合体工作中使用这种杂交鼠胚ES细胞的可能性。     

基于Cre-LoxP系统的条件性定点敲入人源hRas基因小鼠的建立

目的 构建基于Cre- LoxP系统的条件性定点敲入人源hRas基因(c-Ha-ras)的小鼠,以获得hRas基因在特定组织器官条件性表达的小鼠模型,用于药物的临床前致癌性安全评价及相关机制研究。方法 首先构建hRas打靶载体,电击法转染入小鼠胚胎干细胞(ES细胞),用正负法筛选阳性ES细胞,通过PCR、 Southern鉴定后,将正确重组hRas基因的ES细胞导入C57BL/6 J小鼠囊胚,移入同步发育的受体鼠子宫,妊娠足月出生的嵌合体小鼠与C57BL/6 J小鼠交配获得杂合子hRasfl/+小鼠。再将杂合子hRasfl/+小鼠间交配获得纯合子小鼠hRasfl/fi,然后与全身组织细胞表达Cre重组酶的Tg (EIIa-cre)小鼠进行交配,获得全身细胞表达hRas基因的hRas-EIIa-cre小鼠,并通过荧光定量PCR方法检测不同日龄胚胎期仔鼠的hRas基因表达水平。结果 成功构建了基于Cre- LoxP系统的人源hRas基因条件性定点敲入小鼠模型的载体,筛选得到的一个正确克隆,电转ES细胞后进行Southern blot鉴定,经过初筛和复筛,共获得12个阳性克隆,挑选A11号克隆ES细胞进行囊胚注射,移植了48枚胚胎,出生9只小鼠,其中6只为嵌合鼠,将嵌合率>50%的雄鼠与野生型C57BL /6 J雌鼠进行交配,出生21只后代,其中鉴定有4只hRasfl/+小鼠;hRasfl/+小鼠间交配出生29只小鼠,其中有14只纯合子hRasfl/fi小鼠;hRasfl/fl小鼠与Tg (EIIa-cre)工具鼠交配6次,未有仔鼠出生;跟踪不同发育时期胚胎中hRas基因的表达发现,E10.5～15.5 d胚胎中检测到hRas基因表达。结论 成功建立运用Cre- LoxP系统建立了带有hRas基因敲入的纯合子小鼠hRasfl/fl,未能得到全身细胞高效表达人源hRas基因的hRas-EIIa-cre小鼠,但为进一步利用hRasfl/fl小鼠模型建立其他组织特异性的条件性基因敲入小鼠模型做好技术储备。     

血管内皮生长因子C嵌合体（CA65）转基因小鼠的建立和分析

将血管内皮生长因子C嵌合体CA65插入角蛋白14（keratin 14,K14）启动子下游,构建了嵌合体CA65转基因表达载体,通过显微注射法建立转基因小鼠.利用特异引物聚合酶链式反应法鉴定转基因小鼠的基因型后,通过逆转录聚合酶链式反应检测嵌合体CA65的表达水平,证实成功建立了皮肤特异表达嵌合体CA65转基因小鼠.对...     

基于Cre/Loxp系统的Ankle2 基因敲除鼠模型的建立

建立Ankle2基因敲除小鼠模型,为研究该基因在小鼠体内所发挥的重要生理功能提供基础. 本实验利用条件性基因敲除技术,进行打靶载体的设计与构建.将LoxP1st插入到Ankle2基因的第二内含子里,在第五内含子里插入FRT-neo-FRT-LoxP 元件,利用限制性内切酶DNA 及Southern blot 筛选出中靶ES 细胞克隆,随后将发生同源重组的ES细胞注射进C57BL/6J小鼠囊胚中,移入受体小鼠子宫,将得到的嵌合体雄鼠与C57BL/6J雌鼠交配获得Ankle2-Floxed 小鼠. 随后将Ankle2-Floxed 小鼠与全身表达FLP 酶的小鼠进行杂交,将打靶载体中的Neo基因去除,获得F1 代小鼠,F1 代小鼠自交并经PCR 鉴定筛选出Ankle2flox/flox小鼠.Ankle2flox/flox小鼠与全身或组织特异性表达Cre酶小鼠进行杂交,得到Ankle2基因杂合敲出小鼠,该小鼠自交,可获得Ankle2基因纯合敲除的小鼠模型. 该模型为深入研究Ankle2 基因在胚胎发育和衰老发生等中的调控功能提供材料和思路.     

小鼠体细胞核移植及ES细胞样集落分离

利用小鼠皮肤成纤维细胞为核供体进行体细胞核移植并从重构胚中分离胚胎干细胞(ES)样集落,以便对体细胞核移植重构胚来源的ES细胞样集落进行研究.结果显示,小鼠皮肤成纤维细胞作为核供体,核移植重构胚激活率为60.48%(254/420),囊胚发育率为6.90%(29/420),6个囊胚中分离出ES细胞样集落,分离率为1.43%(6/420),3个ES细胞样集落能够稳定传代,至第5代时核型正常率分别为77.84%,75.18%,77.20%.分离出的ES细胞样集落具有岛屿状团状隆起结构,碱性磷酸酶染色呈阳性,体外可自发分化成上皮样或梭形细胞.实验证实小鼠唇部皮肤成纤维细胞能够支持体细胞核移植重构胚发育至囊胚,并能分离出可以稳定传代的ES细胞样集落.     

小鼠胚胎干细胞对病毒性心肌炎的影响

目的使用小鼠验证这样一个假设:外界病毒浸入诱发心肌炎时,机体的干细胞将进入心脏提高心肌的抗病毒能力。方法雄性BALB/c小鼠分为三组:小鼠胚胎干细胞对照组(ES),心肌炎病毒组(EM CV)及EM-CV加ES治疗组。通过尾静脉注射,令小鼠立即感染病毒。小鼠死亡率,炎性细胞浸润及心肌坏死等为观察指征。干细胞的游走及分化等通过免疫荧光法来验证。结果给予干细胞后的小鼠的存活率明显高于生理盐水对照组,炎性细胞侵润及心肌坏死亦明显低于生理盐水对照组。免疫荧光法表明,干细胞进入心肌并分化成新的心肌细胞。结论干细胞能明显提高心肌炎小鼠的存活率,减少心肌组织的坏死。同时,亦证明当心脏遭受病毒的侵入后,干细胞通过某种机理修复或再生心肌细胞,从而提高组织的抗病毒能力。     

用胚泡注射法产生小鼠嵌合体

用免疫手术法分离小鼠胚泡的内细胞团,然后通过显微操作把内细胞团注入另一品系小鼠的胚泡腔内,经体外培养和移植到受体,生下了嵌合体小鼠.其方法是将淡棕色小鼠[(昆明白×C_(57))杂种自交20代以上后培育成的]或昆明白小鼠的胚泡置于0.5%链霉旦白酶中去透明带,然后把裸胚放入1:4稀释的兔抗鼠血清中去滋胚层。抗血清是连续5周,每周一次静脉注射小鼠脾     

纳米银在小鼠体内的组织分布

将分散在生理盐水中的纳米银以尾静脉注射的方式注入小鼠体内,采用电感耦合等离子质谱(ICP-MS)法检测其在小鼠体内的组织分布情况,并利用DAS药物动力学软件分析其在血液中的动力学参数.实验结果显示:纳米银经尾静脉注射进入小鼠体内后,血液中的w(Ag)在染毒1 h内呈快速下降趋势,其后的24 h内w(Ag)处于平稳状态;纳米银在体内的消除半衰期为22.8 h,分布容积为26.8 L/kg;进入体内的纳米银可随血液循环快速分布到全身各脏器,6 h后即可在小鼠的多个主要脏器中检测到银的存在,其中脾脏中的w(Ag)约为203μg/g;滞留在体内的银主要分布在肝脏和脾脏中.由此可见,纳米银具有较高的组织亲和力,肝脏和脾脏可能是纳米银的主要蓄积作用靶器官.     

Hypervariable ultra-long telomeres in mice

Telomere structure and behaviour is less well understood in vertebrates than it is in ciliates and yeasts (reviewed in ref. 1). Like all other eukaryotic chromosomes, those of vertebrates terminate in an array of a short repeated sequence. In vertebrates this sequence is (TTAGGG)n, as shown by in situ hybridization. In humans, these terminal repeats are heterogeneous in length, averaging about 10 kilobases in blood cells. Here we report the structure and inheritance of the terminal repeats present at mouse telomeres. The (TTAGGG)n tracts are many times larger than those present at human telomeres. Because of their constancy in length through somatic cell divisions, they are resolved as multiple discrete restriction fragments of up to 150 kilobases. Strikingly, this banding pattern is highly polymorphic within populations of inbred mice, suggesting an unusually high mutation rate. Indeed, although the banding pattern is inherited in a largely mendelian fashion, (TTAGGG)n tracts of new size appear frequently in family studies.     

Morphine reward in dopamine-deficient mice

Dopamine has been widely implicated as a mediator of many of the behavioural responses to drugs of abuse. To test the hypothesis that dopamine is an essential mediator of various opiate-induced responses, we administered morphine to mice unable to synthesize dopamine. We found that dopamine-deficient mice are unable to mount a normal locomotor response to morphine, but a small dopamine-independent increase in locomotion remains. Dopamine-deficient mice have a rightward shift in the dose-response curve to morphine on the tail-flick test (a pain sensitivity assay), suggesting either a decreased sensitivity to the analgesic effects of morphine and/or basal hyperalgesia. In contrast, dopamine-deficient mice display a robust conditioned place preference for morphine when given either caffeine or l-dihydroxyphenylalanine (a dopamine precursor that restores dopamine throughout the brain) during the testing phases. Together, these data demonstrate that dopamine is a crucial component of morphine-induced locomotion, dopamine may contribute to morphine analgesia, but that dopamine is not required for morphine-induced reward as measured by conditioned place preference.     

Transgenic mice with inducible dwarfism

The pituitary gland, composed of the anterior, intermediate and posterior lobe, represents a principal regulatory interface through which the central nervous system controls body physiology. The ontogeny of the growth hormone (GH) and prolactin (Prl) producing cells of the anterior pituitary has been analysed in transgenic mice, using the thymidine kinase obliteration system (TKO). Cells expressing the herpes virus 1 thymidine kinase (HSV1-TK) gene acquire pharmacological sensitivity to synthetic nucleosides such as FIAU (1-(2-deoxy-2-fluoro-beta-delta-arabinofuranosyl)-5-iodouracil), whose metabolites kill dividing cells. Consequently we created transgenic mice carrying the HSV1-TK gene under the control of either the rat growth hormone or the rat prolactin promoter. If transgenic mice expressing HSV1-TK in somatotropes (GH-producing cells) are treated with FIAU, they develop as dwarfs. The anterior pituitary in these animals is nearly devoid of both somatotropes and lactotropes (Prl-producing cells). By contrast, transgenic mice expressing HSV1-TK in the lactotropes, treated with FIAU, have anatomically and histologically normal pituitaries. Because toxicity depends on cell division, we conclude that Prl expression and lactotrope differentiation are post-mitotic events. These results indicate that both somatotropes and lactotropes derive from a common GH-expressing stem-somatotrope. Unexpectedly, the stemsomatotrope is still present in the adult animal and is capable of repopulating the pituitaries of treated animals with mature GH and Prl producing cells.     

RNA interference in adult mice

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.     

Pre-B cells in kappa-transgenic mice

Recent experiments have shown that the microinjected kappa-chain gene of transgenic mice is expressed in a tissue-specific fashion only in B lymphocytes. The next step was to determine whether, within the B-lymphocyte lineage, the kappa-chain gene was expressed in a normal developmental fashion. Normally, only mu heavy(H)-chain genes, and not kappa-chain genes, are expressed in pre-B cells. To obtain cloned cell lines derived from early cells of the B-cell lineage, we transformed bone marrow cells from kappa-transgenic mice with Abelson murine leukaemia virus (A-MuLV) and tested the resultant cell lines for the retention of the kappa transgene and its expression in RNA and protein. We found that cells with the pre-B phenotype exist in kappa-transgenic mice. We further observed that in A-MuLV-transformed cell lines from a kappa-transgenic mouse with a high copy number of the transgene, the proportion of cell lines expressing kappa (transgenic kappa) was higher than in cell lines from normal or low copy number transgenic mice.