活化的肝星状细胞RBP-J可诱导性基因敲除小鼠的构建及应用

目的:利用Cre-loxp可诱导系统构建活化的肝星状细胞RBP-J可诱导性基因敲除小鼠模型,以实现肝纤维化过程中活化的肝星状细胞Notch信号通路的特异性阻断。方法:将Sm22αCre ERT2和RBP-Jflox/flox转基因小鼠杂交,得到F1小鼠,将繁殖的F1小鼠继续进行交配,得到F2小鼠,通过PCR鉴定出基因型为Sm22αCre ERT2,RBP-Jflox/flox的小鼠。通过腹腔注射四氯化碳建立肝纤维化模型,检测四氯化碳注射不同时间段后肝脏纤维化进展情况和肝星状细胞活化过程中Sm22α的表达情况。注射他莫昔芬诱导活化肝星状细胞中RBP-J基因的特异性敲除。分选肝星状细胞,q-PCR和Western Blot检测活化肝星状细胞中Notch下游靶基因Hes1、Hey1表达情况以验证敲除效果。结果:通过连续交配我们获得了Sm22αCre ERT2,RBP-Jflox/flox小鼠。四氯化碳腹腔注射4周即可诱导肝脏发生较为明显的纤维化,同时肝星状细胞活化并逐渐高表达Sm22α。给予他莫昔芬诱导Cre重组酶发挥作用后,敲除了活化的肝星状细胞中的RBP-J基因,其下游基因Hes1、Hey1表达降低70%以上,阻断了Notch信号通路。结论:我们成功构建了RBP-J可诱导性条件性基因敲除小鼠模型,实现了小鼠肝纤维化过程中活化肝星状细胞Notch信号通路的阻断,为深入研究Notch信号通路在肝纤维化中的作用和机制奠定了坚实的基础。     

神经干细胞特异性LSD1基因敲除对小鼠情绪及记忆的影响

目的条件敲除小鼠神经干细胞LSD1基因后,通过观察小鼠神经细胞增殖情况及小鼠行为的表现,揭示LSD1的神经生物学功能。方法将LSD1(flox/flox)转基因小鼠与神经干细胞特异性表达Cre重组酶的Nestin-cre(Tg)转基因小鼠进行的杂交,即利用Cre-Lox P系统,繁殖出LSD1(flox/flox)Nestin-cre(Tg)基因型小鼠,即为所需神经干细胞LSD1条件性敲除小鼠(LSD1-CKO),LSD1(flox/flox)做为对照小鼠。进一步运用免疫荧光染色方法检测小鼠海马DG区神经细胞的增殖;并通过小鼠的糖水偏好、强迫游泳及新物体识别等实验检测小鼠的行为表现。结果与LSD1(flox/flox)小鼠相比,LSD1-CKO小鼠海马区神经细胞增殖显著降低(P=0.023);糖水偏好系数降低(P=0.0075);强迫游泳实验中不动时间明显增加(P0.05);并在新物体识别实验中表现出记忆力显著下降现象(P=0.0019)。结论小鼠脑神经干细胞敲除LSD1基因,海马区神经细胞增殖降低,LSD1-CKO小鼠表现出负面情绪和记忆障碍。     

心肌特异性Creg基因条件敲除小鼠的建立及表型分析

目的:建立心肌特异性Creg基因敲除小鼠并初步分析其表型。方法:利用订购的Creg两端插入lox P位点(Cregflox/flox)的小鼠与肌型肌酸激酶特异性启动子驱动的Cre重组酶转基因(Ckmm-cre)小鼠交配,获得Cregflox/+/Ckmm-cre小鼠。再利用Cregflox/+/Ckmm-cre小鼠互相交配,获得基因型为Cregflox/flox/Ckmm-cre的心肌特异性Creg基因条件敲除(Creg conditional knockout,Creg c KO)小鼠。用PCR法进行基因型鉴定。用定量PCR及Western Blot检测心肌组织中Creg表达水平。HE染色观察敲除小鼠与同窝野生型对照小鼠心脏大小及形态。检测两组小鼠心电图。用小动物超声评价两组小鼠左心室收缩功能。结果:1经基因型鉴定,成功获得Creg c KO小鼠。2与野生型对照相比,Creg c KO小鼠心脏中CREG在转录及翻译水平表达降低90%以上。3与野生型对照相比,Cre c KO小鼠的心脏大小、形态、心电图及左心室射血分数均无显著差别。结论:成功建立心肌特异性CREG基因条件敲除小鼠,为进一步研究Creg在心脏疾病中的作用和机制提供了有力的工具。     

少突胶质细胞条件性敲除FGF9基因小鼠模型的建立、鉴定及初步表型

目的 建立少突胶质细胞特异性敲除成纤维生长因子9(FGF9)小鼠模型,进一步研究FGF9在神经发育中的作用.方法 将Olig1-Cre转基因小鼠与FGF9转基因小鼠(FGF9flox/flox)杂交,选取雌性FGF9flox/wt/Olig1-Cre+与雄性FGF9flox/flox合笼交配,F3代获得少突胶质细胞特异...     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

肾上腺皮质束状带特异性表达Cre重组酶转基因小鼠的构建

肾上腺是人体重要的内分泌器官。由于缺乏肾上腺皮质束状带特异性表达Cre酶的工具鼠,目前对肾上腺皮质束状带细胞中特异表达基因的功能缺乏深入的解析。CYP11B1基因编码类固醇11β-羟化酶,该酶是糖皮质激素合成的关键酶,在肾上腺皮质束状带中特异性表达。本研究旨在利用CYP11B1基因在束状带特异性表达的特点,构建在肾上腺皮质束状带中特异性表达Cre重组酶的转基因动物。采用CRISPR/Cas9技术在CYP11B1基因终止密码子位点定点敲入2A-GfpCre表达框,获得CYP11B1-2A-GfpCre同源重组载体,进而构建CYP11B1Cre小鼠,并通过mTmG和LacZ染色确定Cre酶主要表达在小鼠肾上腺皮质束状带。在此基础上,本研究还用该工具鼠与胱硫醚-γ-裂解酶(cystathionineγ-lyase, CTH)条件性敲除鼠交配,获得了肾上腺皮质束状带CTH特异性敲除的小鼠,并证实了该动物肾上腺皮质束状带中CTH表达缺失。以上结果充分说明肾上腺皮质束状带特异性表达Cre重组酶小鼠构建成功。该工具鼠的成功构建,为深入研究肾上腺皮质束状带相关功能提供了有力工具。     

乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠的制作与鉴定

目的利用Cre．LoxP重组酶系统构建乳腺上皮细胞特异性敲除Serib基因杂合子小鼠,并进行鉴定,为进一步在动物整体水平研究Scrib基因在乳腺癌中的作用提供研究平台。方法将Scrib条件敲除杂合子小鼠（Scrib＋/ft小鼠）进行繁殖并鉴定,然后将鉴定结果为阳性的子代Scrib＋/ft小鼠与乳腺上皮细胞特异性表达Cre重组酶的MMTV．Cre纯合子小鼠进行杂交,鉴定其子代小鼠的基因型。结果成功繁育Scrib条件敲除小鼠和MMTV．Cre小鼠,并通过鉴定得到Scrib＋/ft小鼠,与MMTV-Cre小鼠杂交并繁殖,获得基因型为Scrib＋/ft;MMTVCre＋/-小鼠5只。结论本研究利用Cre．LoxP重组酶系统成功构建了乳腺上皮细胞特异性敲除Scrib基因杂合子小鼠,为进一步研究极性蛋白Scrib表达下调在乳腺癌发生中的作用提供了良好的动物模型。     

成骨细胞特异性表达Cre重组酶转基因小鼠的建立

构建了含有骨钙素基因启动子、Cre重组酶基因和人生长激素基因polyA的转基因载体pOC-Cre, 以显微注射的方法将4.6 kb的转基因片段OC-Cre导入小鼠受精卵。16只子代小鼠中经PCR和Southern杂交鉴定, 有2只小鼠携带外源基因, 整合率为12.5%。为了检测OC-Cre在转基因小鼠中表达的组织特异性, 将转基因首建者小鼠与基因组上携带有LoxP位点的条件性Smad4基因敲除小鼠交配, PCR结果显示, 仅在子代纯合型小鼠骨组织基因组中扩增出了Cre介导重组后的片段。将OC-Cre转基因小鼠与ROSA26报告小鼠交配, 利用LacZ染色对双转基因阳性子代小鼠进行检测, 结果显示Cre重组酶在成骨细胞中特异性表达并介导ROSA基因座LoxP位点间的重组。所有这些结果说明：所建立的OC-Cre转基因小鼠在成骨细胞中特异性表达Cre重组酶, 并能在体内介导成骨细胞基因组上LoxP位点间的重组, 是一种理想的研制成骨细胞特异性基因敲除小鼠的工具小鼠。     

血管内皮细胞条件性敲除Cx43杂合子小鼠模型建立及其血管功能变化的研究

目的 建立血管内皮细胞连接蛋白43(connexin 43, Cx43)条件性敲除小鼠模型,并对其血管舒张/收缩功能进行检测。方法 将8只Cx43^flox/flox 小鼠与C57BL/6品系野生型(wild type, WT)小鼠交配,子代小鼠继续与C57BL/6小鼠回交9代进行基因背景转换;再将Cx43^flox/+小鼠与血管内皮细胞特异性表达Tie2-Cre重组酶小鼠(以下简称Cre小鼠)交配,获得Tie2-Cre/Cx43^flox/+小鼠。利用鼠尾PCR试剂盒进行基因型鉴定,Western Blot法和免疫荧光法检测小鼠肠系膜上动脉(superior mesenteric artery, SMA)中Cx43表达。利用离体张力测定技术检测SMA血管舒张/收缩功能,包括去甲肾上腺素诱导的收缩反应性和乙酰胆碱诱导的舒张反应性。结果 PCR电泳和血管蛋白表达检测结果证实Tie2-Cre/Cx43^flox/+小鼠成功构建,肠系膜上动脉中Cx43蛋白表达与野生型小鼠相比明显降低(P<0.01)。与WT...     

增强子驱动的Cre转基因小鼠的构建及Cre基因的表达鉴定

目的:探索将增强子应用于构建Cre转基因小鼠品系,为以条件基因敲除为基础的基因功能研究提供更多的工具。方法:通过PCR方法从小鼠的细菌人工染色体扩增UH增强子片段,构建含有Hsp68基础启动子、增强子UH、Cre重组酶基因和SV40 polyA的转基因载体pLW400,将3.3 kb的转基因片段通过显微注射导入小鼠受精卵;为了检测Cre在转基因小鼠中的表达,将转基因一代小鼠与纯合子ROSA26报告小鼠(R/R)交配,收集第14 d胚胎期(E14)的舌组织进行LacZ染色检测鉴定。结果:经鉴定,31只子代小鼠中有6只携带外源基因,整合率为19.4%;与R/+对照相比,E14期的双基因型Cre,R/+舌组织为阳性结果(蓝色)。这表明Cre基因在转基因小鼠舌组织内得到表达,并在体内介导ROSA26基因座loxP位点间的重组,且有效删除了2个loxP之间的片段,从而启动了LacZ基因的表达。结论:构建了UH增强子-Hsp68Cre的转基因小鼠,在舌肌中特异表达Cre基因,提示增强子可以被选择应用于Cre转基因小鼠的构建;为舌肌的发育和再生研究奠定了基础。     

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.     

Generation of PECAM‐1 (CD31) conditional knockout mice

Platelet endothelial cell adhesion molecule 1 (PECAM‐1) is an adhesion and signaling receptor that is expressed on endothelial and hematopoietic cells and plays important roles in angiogenesis, vascular permeability, and regulation of cellular responsiveness. To better understanding the tissue specificity of PECAM‐1 functions, we generated mice in which PECAM1, the gene encoding PECAM‐1, could be conditionally knocked out. A targeting construct was created that contains loxP sites flanking PECAM1 exons 1 and 2 and a neomycin resistance gene flanked by flippase recognition target (FRT) sites that was positioned upstream of the 3′ loxP site. The targeting construct was electroporated into C57BL/6 embryonic stem (ES) cells, and correctly targeted ES cells were injected into C57BL/6 blastocysts, which were implanted into pseudo‐pregnant females. Resulting chimeric animals were bred with transgenic mice expressing Flippase 1 (FLP1) to remove the FRT‐flanked neomycin resistance gene and mice heterozygous for the floxed PECAM1 allele were bred with each other to obtain homozygous PECAM1 ^flox/flox offspring, which expressed PECAM‐1 at normal levels and had no overt phenotype. PECAM1 ^flox/flox mice were bred with mice expressing Cre recombinase under the control of the SRY‐box containing gene 2 (Sox2Cre) promoter to delete the floxed PECAM1 allele in offspring (Sox2Cre;PECAM1 ^del/WT), which were crossbred to generate Sox2Cre; PECAM1 ^del/del offspring. Sox2Cre; PECAM1 ^del/del mice recapitulated the phenotype of conventional global PECAM‐1 knockout mice. PECAM1 ^flox/flox mice will be useful for studying distinct roles of PECAM‐1 in tissue specific contexts and to gain insights into the roles that PECAM‐1 plays in blood and vascular cell function.     

Generation of a conditional null allele of Lbx1

The homeobox gene Lbx1 not only plays critical roles in myogenesis and neurogenesis during embryonic development but is also expressed in activated satellite cells of adult mice. To address the potential postnatal functions of Lbx1, we generated conditional Lbx1-null mice using the Cre-loxP system. We generated a mouse in which Exon 2 of Lbx1 was floxed (Lbx1flox/flox), followed by cross-breeding between the Lbx1flox/flox mouse and either a transgenic mouse where a tamoxifen-inducible Cre-recombinase (Cre) was ubiquitously expressed, or a Myf5Cre mouse where Cre was inserted into the Myf5 locus. In both Lbx1-null mouse lines generated, Pax3-expressing limb muscle precursor cells were seriously reduced during embryonic development and eventually the limb extensor muscles were lost after birth. Since the conditional Lbx1-null mice generated were viable for a prolonged time, they will be useful in the investigation of Lbx1 function throughout the lifespan of the mouse.     

Dkk3-Cre BAC transgenic mouse line: a tool for highly efficient gene deletion in retinal progenitor cells

To establish the genetic tools for conditional gene deletion in mouse retinal progenitors, we generated a Dkk3-Cre transgenic mouse line using bacterial artificial chromosome (BAC) transgenesis. Cre recombination efficiency in vivo was assayed by crossing this transgenic line, termed BAC-Dkk3-Cre, with the CAG-CAT-Z reporter line. This BAC-Dkk3-Cre line showed Cre recombinase activity in most retinal progenitors. Cre activity was detectable from embryonic day 10.5 (E10.5) and generally restricted to the retina during embryogenesis. To verify that BAC-Dkk3-Cre mice successfully circumvented lethality, we generated Otx2flox/flox/BAC-Dkk3-Cre+ mice as Otx2 conditional knockout mice. The Otx2flox/flox/BAC-Dkk3-Cre+ mice were viable, and their retina showed loss of mature cell-type markers of photoreceptor cells, bipolar cells, and horizontal cells, in contrast, amacrine-like cells noticeably increased. Thus, the BAC-Dkk3-Cre transgenic mouse line provides a powerful tool for generating conditional knockout mouse lines for studying loss of gene functions in the developing retina.     

Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting β-cells, we have generated mice with a β-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre(+/-) Dicer1(Δ/wt)), RIP-Cre(+/-)Dicer1(flox/flox) mice (RIP-Cre Dicer1(Δ/Δ)) developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased β-cell mass, reduced numbers of granules within the β-cells and reduced granule docking in adult RIP-Cre Dicer1(Δ/Δ) mice. β-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal β-cell development as 2-week old RIP-Cre Dicer1(Δ/Δ) mice showed ultrastructurally normal β-cells and intact insulin secretion. In conclusion, we have demonstrated that a β-cell specific disruption of the miRNAs network, although allowing for apparently normal β-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.     

A transgenic Cre mouse line for the study of cortical and hippocampal development

Wnt signaling regulates cortical and hippocampal development. In a previous study we found that a particular Wnt receptor, Frizzled9 (Fzd9), was selectively expressed in both the developing and adult hippocampus. Taking advantage of the specificity of this promoter, we generated a transgenic cre mouse line using the putative control elements of the Fzd9 gene. In the Fzd9‐cre mice, Cre is mainly detected in the developing cortex and hippocampus and is confined to the CA fields and dentate gyrus in adults. Furthermore, by crossing the Fzd9‐cre mouse with the ROSA26 reporter line, we examined the activity of Cre and found that it has very high recombination efficiency. Thus, this mouse line will likely prove to be a useful tool for studying cortical and hippocampal development via activation or inactivation of interesting genes. genesis 48:343–350, 2010. © 2010 Wiley‐Liss, Inc.     

红景天苷对癫痫大鼠认知功能障碍的治疗作用及其可能机制

目的：探讨红景天苷（sal）对癫痫大鼠认知功能障碍的治疗作用及其可能机制。方法：将24只成年雄性SD大鼠随机分为健康对照组、模型组、Sal[按体重1g／（kg·d）]干预组。采用Morris水迷宫方法检测大鼠学习记忆功能变化,并检测大鼠脑组织中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH—PX）和谷胱甘肽（GSH）、丙二醛（MDA）相应的比酶活力及含量变化。结果：（1）模型组大鼠寻找平台的潜伏期明显长于对照组,具有统计学意义（P〈0．05）,Sal组寻找平台的潜伏期相对于模型组显著缩短（P〈0．05）。撤离平台后,模型组大鼠在平台所在象限的停留时间明显短于对照组（P〈0．05）,sal治疗后大鼠在平台所在象限的停留时间较模型组显著延长（P〈0．05）。（2）模型组SOD、GSH、GSH—Px显著下降,MDA明显增高,Sal干预组SOD、GSH、GSH—PX明显增高．而MDA显著下降,有统计学差异（P〈0．05）。结论：Sal可减轻癫痫持续状态所致的认知功能障碍,其可能机制是通过减轻海马区氧化应激减轻海马区的损伤,进而改善认知功能。