基因敲除小鼠技术的研究进展

基因敲除小鼠技术的建立和发展使得人们为研究基因的功能和寻找新的治疗人类疾病的靶点提供了强有力的支持。基因打靶和基因捕获是两种通过胚胎干细胞(Embryonicstemcell,ESC)构建基因敲除小鼠的技术。基因打靶通过同源重组替换内源基因从而敲除目的基因,而基因捕获则有启动子捕获和polyA捕获两种方法对目的基因进行敲除。近年来,有许多新的基因敲除技术不断被开发出来,包括Cre/loxP系统、CRISP/Cas9系统以及最新的ZFN技术和TAILEN技术,都有望取代传统基因敲除手段。文中简要阐述了如今新出现的几种基因敲除小鼠技术。     

应用CRISPR/Cas9技术构建YOD1基因敲除小鼠

目的:应用CRISPR/Cas9技术构建去泛素化酶YOD1基因敲除小鼠。方法:针对YOD1基因设计单链向导RNA(sg RNA)识别序列,构建sg RNA质粒,与Cas9质粒体外转录、纯化后注射入受精卵,通过PCR和测序验证得到F0代阳性小鼠。配繁两代后,取同窝对照的野生型(WT)和敲除(KO)小鼠的主要组织器官研磨,使用免疫印迹(WB)技术检测各组织YOD1蛋白的表达,确证YOD1敲除小鼠模型是否成功建立。统计YOD1杂合子(HET)自交存活后代各基因型比例,分析是否有胚胎致死表型。解剖小鼠分析主要组织器官的表型,进一步利用H.E.染色分析KO小鼠是否存在自发的病理改变。通过血糖耐受实验(GTT)分析KO小鼠的血糖调控能力。结果:基因组测序和WB检测结果显示KO小鼠中YOD1被明显敲除,YOD1敲除小鼠模型成功建立。YOD1杂合子自交后代各基因型比例符合孟德尔定律,提示KO小鼠非胚胎致死。YOD1敲除小鼠肝脏显著小于WT小鼠。GTT结果表明敲除YOD1不影响小鼠的血糖稳态。结论:应用CRISPR/Cas9技术成功构建YOD1基因敲除小鼠。KO小鼠正常出生,无任何胚胎发育缺陷。与WT小鼠相比,KO小鼠肝脏显著减小,但无显著的自发病理变化,KO小鼠血糖控制亦无显著差异。     

内皮和造血系统特异性敲除Rictor基因对胎肝造血的影响

目的:研究Rictor基因对胚胎发育过程中胎肝造血的影响。方法:利用Cre-LoxP基因敲除系统,特异性在小鼠内皮(VEC-Cre)和造血(Vav1-Cre)系统敲除Rictor基因;通过流式细胞术分析特异敲除Rictor基因后小鼠胚胎第15 d胎肝中的各系细胞比例的变化,并进一步分析造血干细胞的变化。结果:利用VEC-Cre和Vav1-Cre小鼠敲除Rictor基因后,胎肝中各系细胞比例均有所减少,B细胞比例的减少较为明显,造血干细胞比例也明显减少。结论:Rictor基因敲除损害胎肝组织中造血干细胞的产生和各系细胞的分化。     

胸腺上皮细胞特异性敲除GSK-3β基因小鼠模型的鉴定及免疫学特征观察

目的 构建并鉴定胸腺上皮细胞(thymic epithelial cells, TECs)特异性敲除GSK-3β基因小鼠模型,评价其免疫学特征。方法 采用小鼠胚胎干细胞(ES细胞,embryonic stem cells)打靶基因敲除技术,得到F0代的GSK-3β^flox/flox(GSK-3β^f/f)小鼠;再采用Cre-LoxP系统进行小鼠的繁殖;PCR鉴定,筛选出基因型为GSK-3β^f/fFoxN1-Cre^+/-(GSK-3β^-/-)小鼠,即为在TECs特异性敲除GSK-3β基因的小鼠。观察基因敲除鼠的一般生物学特征、繁殖能力和子代存活率。应用HE染色、流式细胞术及免疫荧光技术,比较基因敲除鼠和野生型小鼠免疫器官结构、胸腺、脾及外周血中免疫细胞比例及增殖能力差异。结果 成功构建TECs特异性敲除GSK-3β基因小鼠模型。与野生型(wild type, WT)小鼠相比,GSK-3β^-/-小鼠一般生物学特征无明显差异,子代存活率> 90%;衰老进程中...     

FKBP52对小鼠前列腺发育的影响

目的通过FKBP52基因敲除小鼠模型探索FKBP52在小鼠前列腺发育过程中的作用。方法分别对胚胎第17．5天、新生的和出生后3周的野生型和FKBP52基因敲除小鼠的前列腺进行切片HE染色,观察不同发育时期里野生型和FKBP52基因敲除小鼠前列腺发育的异同。结果（1）小鼠前列腺发育的起始不依赖于FKBP52基因的参与;（2）随着胚胎的发育,FKBP52在雄鼠前列腺发育中的作用逐渐显现出来,即FKBP52的缺失会导致前列腺叶发育受阻,最终不能形成成熟的前列腺。结论FKBP52在小鼠前列腺的发育过程中具有重要作用,它不参与前列腺的发育起始过程,但其缺失会导致前列腺发育受阻,即不能形成成熟的前列腺。     

Bmal1时钟基因敲除小鼠的繁育及基因型鉴定

目的对Bmal1基因敲除小鼠进行繁育及基因型进行鉴定,为生物节律研究提供理想的动物模型。方法将引进的Bmal1基因敲除小鼠,以1雄2雌的合笼方式进行饲养繁殖,从仔鼠中提取鼠尾基因组DNA,PCR扩增目的基因片段,琼脂凝胶电泳进行基因结果判定,Western Blot检测心肌组织中Bmal1蛋白表达进行结果验证。结果 Bmal1~(-/-)小鼠与Bmal1~(+/+)小鼠相比,表型除体重外未见明显差异,纯合敲除小鼠丧失繁殖能力; Bmal1~(-/-)小鼠24 h血糖节律改变,Bmal1基因敲除小鼠繁育成功,获得一批基因敲除鼠。结论应用PCR法可成功鉴定Bmal1基因敲除纯合小鼠,PCR扩增法是检测小鼠基因的有效方法。     

FKBP38肝脏特异敲除小鼠模型的构建

目的:构建FKBP38(FK506 Binding Protein 38)基因肝脏特异敲除小鼠。方法:利用胚胎注射法构建在FKBP38上携带lox P位点的转基因小鼠。在FKBP38基因位置携带lox P位点的小鼠的基础上,以肝脏实质细胞特异性表达的Alb-Cre介导FKBP38条件性敲除,以获得FKBP38基因肝脏特异敲除小鼠模型Alb-Cre:FKBP38~(fl/fl)。同时对FKBP38特异性敲除鼠进行鉴定。结果:(1)FKBP38肝脏特异敲除小鼠FKBP38~(-/-)肝脏中FKBP38基因的m RNA水平相对于同年龄同窝野生型小鼠具有统计学差异(P0.001)。(2)FKBP38肝脏特异敲除小鼠FKBP38~(-/-)肝脏中FKBP38基因的蛋白表达水平相对于同年龄同窝野生型小鼠具有统计学差异(P0.001)。(3)FKBP38肝脏特异敲除小鼠FKBP38~(-/-)肝脏中,转录和翻译相关蛋白水平未见显著差异,p70 S6K的磷酸化水平轻微上调,4EBP-1的磷酸化水平有轻微下调。(4)FKBP38肝脏特异敲除小鼠FKBP38~(-/-)肝脏中,凋亡相关蛋白Bcl-2未见差异化表达。结论:FKBP38肝脏特异敲除小鼠FKBP38~(-/-)肝脏中,FKBP38基因的m RNA和蛋白基本不表达,提示成功构建FKBP38基因肝脏特异敲除小鼠。     

去泛素化酶OTUB1肝脏特异性基因敲除小鼠模型的构建与表型分析

目的:建立OTUB1肝脏特异性基因敲除小鼠模型,初步分析其表型并研究OTUB1基因与肝脏代谢的关系。方法:利用Cre/Loxp系统构建条件性基因敲除小鼠模型,即将OTUB1~(fl/fl)转基因小鼠与Alb-Cre小鼠杂交,子代自交,得到OTUB1肝特异性基因敲除小鼠并进行鉴定。取同窝对照小鼠(control,NC)和肝特异型基因敲除(hepatic-specific OTUB1 knockout,HCKO)小鼠,通过PCR和免疫印迹(Western blot),确证OTUB1肝脏特异性基因敲除小鼠模型是否成功构建。通过组织病理学方法,分析主要组织器官的形态以及是否存在自发的病变;通过血清生化指标检测肝脏脂代谢水平;通过血糖耐受实验(GTT)分析HCKO小鼠对血糖的控制。结果:基因组测序和Western blot检测结果显示HCKO小鼠肝脏中OTUB1被敲除,其他组织中OTUB1表达水平无变化,证明OTUB1肝脏特异性基因敲除小鼠模型构建成功。HCKO小鼠出生正常,各组织器官无异常,生化指标中总胆固醇水平明显降低,表明OTUB1影响肝脏脂代谢水平。糖耐受实验中HCKO小鼠血糖回落迅速,表明敲除OTUB1影响肝脏血糖调节稳态。结论:应用Cre/Loxp技术成功建立OTUB1肝脏特异性基因敲除小鼠模型,为研究OTUB1在肝脏的生理功能和调控机制提供了重要的动物模型。     

Grx-1基因敲除鼠的繁殖及子代基因型鉴定

目的探讨glutaredoxin-1(Grx-1)基因敲除小鼠的优化繁殖及子代鼠的鉴定方法,为进一步研究Grx-1在支气管肺发育不良(BPD)中的作用奠定基础。方法将从美国哈佛医学院引进的纯合子Grx-1基因敲除小鼠与野生型小鼠进行交配后得到的子一代小鼠同代间相互交配,繁殖出的子二代中将出现纯合子、杂合子以及野生型3种基因型。从出生起观察其生长发育情况,2周龄时剪尾提取基因组DNA,用PCR方法扩增目的基因片段,琼脂糖凝胶电泳结果判定基因型。结果 Grx-1纯合子小鼠的饲养繁殖取得成功,获得了一批Grx-1基因敲除纯合子小鼠。结论正确的饲养繁殖以及鉴定方法是获得Grx-1基因敲除纯合子小鼠的有效途径,为相关研究提供动物实验模型奠定了基础。     

GPR126条件性基因敲除载体的快速构建

基因敲除小鼠模型是在动物体内研究基因功能的重要和可靠的手段之一,而构建用于同源重组的基因敲除载体是构建基因敲除小鼠模型的关键一步.条件性基因敲除技术将针对靶基因的修饰作用限制在小鼠的特定发育时期或特定类型的组织细胞中,通过加入具有位点特异性的重组系统,该修饰作用在时间和空间上均处于可调控的状态;条件性基因敲除技术既可以避免某些具有重要功能基因的敲除所带来的早期胚胎致死问题,还可用于精确分析某些多效基因在不同组织或不同发育阶段的特定功能差异.通过合理利用改良的Red重组系统,将两个LoxP位点快速准确地插入到了目标位置,实现了GPR126条件性基因敲除载体的快速构建,为后续的构建GPR126基因敲除小鼠模型、在动物体内研究基因的功能奠定了基础.     

Lats2 is an essential mitotic regulator required for the coordination of cell division

Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.     

Trinucleotide repeat containing 6a (Tnrc6a)-mediated microRNA function is required for development of yolk sac endoderm

Tnrc6 family members (Tnrc6a/b/c) are key components of the RNA-induced silencing complex in microRNA (miRNA)-mediated gene suppression. Here, we show that Tnrc6a, also known as GW182, is selectively expressed in the yolk sac endoderm and that gene trap disruption of GW182 leads to growth arrest and apoptosis. We found that targets of miRNAs highly expressed in the yolk sac are significantly derepressed in GW182(gt/gt) mutant mice, although levels of miRNAs are not altered. Specifically, growth arrest and apoptosis phenotype are associated with significant derepression of Cdkn1a (p21), Cdkn1c (P27), Lats1, Lats2, Rb1, Rbl, Bim, and Pten, known targets of miRNAs from miR-17/20/93/106 clusters highly expressed in yolk sac endoderm. Together, these data strongly suggest that GW182 is an essential functional component in the RNA-induced silencing complex for miRNA-mediated gene silencing in vivo, and selectively regulation of miRNA activity plays an important role in the proper development of yolk sac.     

Generation of mice with hepatocyte-specific conditional deletion of sphingosine kinase 1

SphK1 gene has different roles in various types of cells in liver diseases, but most studies are based on global knockout mice, which hampers the study on the cellular and molecular mechanisms of SphK1. In order to further study the role of SphK1 in liver, SphK1 conditional knockout mice were constructed. A liver-specific SphK1 gene knockout mouse model was constructed by the Cre/Loxp recombinant enzyme system. PCR technologies and western blotting were used to identified the elimination of SphK1 gene in hepatocytes. SphK1^flox/flox mice were used as a control group to verify the effectiveness of SphK1 liver-specific knockout mice from the profile, pathology, and serology of mice. The ablation of SphK1 in hepatic parenchymal cells was demonstrated by fluorescent in situ hybridization and the contents of S1P and Sph were measured by ELISA kit. The genotypes of liver in SphK1 conditional knockout mice were different from that of other organs. The mRNA and protein levels of SphK1 in liver tissue of SphK1 conditional knockout mice were almost depleted by compared with SphK1^flox/flox mice. Physiology and pathology showed no significant difference between SphK1 liver conditional knockout mice and SphK1^flox/flox mice. Additionally, SphK1 was eliminated in hepatocytes, leading to the reduce of S1P content in hepatocytes and liver tissues and the increase of Sph content in hepatocytes. The model of SphK1 gene liver conditional knockout mice was successfully constructed, providing a tool for the study of the roles of SphK1 in hepatocyte and liver diseases.

     

Muc2和DCN基因敲除小鼠食子现象的初步研究

目的探讨Muc2和DCN基因敲除小鼠繁殖能力和食子现象的异同。方法分别将Muc2和DCN基因敲除纯合子雌雄小鼠按1∶1或1∶2的合笼,观察1～3胎产仔量、胎次间隔时间、出生存活率和食子现象。结果Muc2基因敲除小鼠平均产子量5.80±0.95只,平均胎次间隔时间（42.29±2.28）d;DCN基因敲除小鼠平均产子量3.85±0.76只,平均胎次间隔时间（24.86±10.42）d。Muc2和DCN基因敲除小鼠在产仔量、胎次间隔时间、出生存活率和食子率差异均存在显著性。结论两组基因敲除小鼠繁殖性能有差异,揭示可能与Muc2和DCN基因有关。     

转录因子Egr-1参与长期性恐惧记忆和焦虑

锌指转录因子点Egr-1在将细胞外信号和胞内基因表达的变化相耦联过程中发挥重要的作用。海马和杏仁体是记忆形成和储存的两个主要的脑区。在海马和杏仁体中,Egr-1可被长时程增强（long-term potentiation,LTP）和学习过程上调。在Egr-1敲除小鼠上观察到晚时相声音恐惧记忆受损,而短时的痕迹和场景记忆却不受影响;另外,在Egr-1敲除小鼠上,用theta burst刺激杏仁体和听觉皮层所引起的突触增强被明显减弱或完全阻断。因此,我们的研究表明,转录因子Egr-1选择性地在晚时相听觉恐惧记忆中发挥作用。     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.     

Exo-1基因对端粒酶缺陷小鼠造血干细胞植入效率的影响

目的利用不同的造血干细胞移植方式,探讨Exo-1基因缺失对端粒酶基因敲除小鼠的造血干细胞植入效率的影响。方法以CD45.1小鼠的骨髓细胞或骨髓造血干细胞为供体,以端粒酶基因敲除小鼠或Exo-1基因和端粒酶基因双敲除小鼠为受体,在给予不同剂量X线照射或不照射的情况下,重复进行静脉注射全骨髓细胞或分选的骨髓造血干细胞(c-kit+、Sca-1+、lineage-,KSL),于移植后1个月取外周血,流式分析嵌合率。结果未经X线照射及1 Gy、2 Gy照射情况下,端粒酶基因缺陷受体小鼠的外周血中供体来源的细胞嵌合率较低;6 Gy照射后,供体来源的外周血细胞嵌合率仍低于50%,而且端粒酶基因缺陷受体小鼠在移植后1个月内死亡较多;3 Gy照射可形成较高嵌合率,Exo-1基因缺失对端粒酶缺陷小鼠的造血干细胞植入效率的影响不显著。结论以端粒酶缺陷小鼠作为衰老模型研究造血干细胞植入效率时,3 Gy X线照射能够有效地形成较高的外周血供体细胞的嵌合率,但是Exo-1基因没有进一步提高造血干细胞在端粒酶敲除小鼠的植入效率。     

The tumor suppressor Lats2 is pivotal in Aurora A and Aurora B signaling during mitosis

Accurate coordination between chromosome segregation and cytokinesis by various mitotic kinases, such as Aurora, prevent tetraploidization and subsequent tumorigensis. The tumor suppressors Lats1 and Lats2 are serine/threonine kinases that localize to the centrosome and regulate cell cycle progression and apoptosis. In the present study, Aurora A was demonstrated to phosphorylate Lats2 on serine 380 (S380) during mitosis. Immunocytochemical observations revealed that the subcellular localization of Lats2 was distinct during the cell cycle and depended on which site was phosphorylated. Interestingly, the S380-phosphorylated Lats2 protein (pS380) colocalized at the central spindle with Aurora B. Physical interactions were observed between Aurora A, Lats2, Lats1 and Aurora B. The Lats1 kinase was shown to phosphorylate Aurora B. Cells expressing a nonphosphorylated mutant (S380A) of Lats2 caused chromosome missegregation and cytokinesis failure, similar to cells with aberrantly expressed Aurora B. Together, the results suggest that the Aurora A-Lats1/2-Aurora B axis might be a novel pathway that regulates accurate mitotic progression by ensuring the proper mitotic localization of Lats2.     

Increased methamphetamine-induced locomotor activity and behavioral sensitization in histamine-deficient mice

We have recently suggested that the brain histamine has an inhibitory role on the behavioral effects of methamphetamine by pharmacological studies. In this study, we used the histidine decarboxylase gene knockout mice and measured the spontaneous locomotor activity, the changes of locomotion by single and repeated administrations of methamphetamine, and the contents of brain monoamines and amino acids at 1 h after a single administration of methamphetamine. In the histidine decarboxylase gene knockout mice, spontaneous locomotor activity during the dark period was significantly lower than in the wild-type mice. Interestingly, methamphetamine-induced locomotor hyperactivity and behavioral sensitization were facilitated more in the histidine decarboxylase gene knockout mice. In the neurochemical study, noradrenaline and O-phosphoserine were decreased in the midbrain of the saline-treated histidine decarboxylase gene knockout mice. On the other hand, single administration of methamphetamine decreased GABA content of the midbrain of the wild-type mice, but did not alter that of histidine decarboxylase gene knockout mice. These results suggest that the histamine neuron system plays a role as an awakening amine in concert with the noradrenaline neuron system, whereas it has an inhibitory role on the behavioral effects of methamphetamine through the interaction with the GABAergic neuron system.