Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult hom...Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.展开更多
BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remai...BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.展开更多
Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and ...Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.展开更多
Mouse models of cancer enable researchers to learn about tumor biology in complicated and dynamic physiological systems. Since the development of gene targeting in mice, cancer biologists have been among the most freq...Mouse models of cancer enable researchers to learn about tumor biology in complicated and dynamic physiological systems. Since the development of gene targeting in mice, cancer biologists have been among the most frequent users of transgenic mouse models, which have dramatically increased knowledge about how cancers form and grow. The Chinese Journal of Cancer will publish a series of papers reporting the use of mouse models in studying genetic events in cancer cases. This editorial is an overview of the development and applications of mouse models of cancer and directs the reader to upcoming papers describing the use of these models to be published in coming issues, beginning with three articles in the current issue.展开更多
The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,al...The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,although it has been popular during the past decades.Recent years,genome editing which can cause DNA sequence-specific mutations in the genomes of cellular展开更多
背景:对于3-磷酸肌醇依赖性蛋白激酶1(3-phosphoinositide-dependent protein kinase 1,PDK1)的学术研究,国内外主要集中在内分泌和肿瘤学等学科领域,而在骨科学中关于调控破骨细胞对骨质疏松的改善尚未有系统研究与报道。目的:探索PDK...背景:对于3-磷酸肌醇依赖性蛋白激酶1(3-phosphoinositide-dependent protein kinase 1,PDK1)的学术研究,国内外主要集中在内分泌和肿瘤学等学科领域,而在骨科学中关于调控破骨细胞对骨质疏松的改善尚未有系统研究与报道。目的:探索PDK1调控破骨细胞对骨质疏松症发病的影响及分子机制,为临床防治骨质疏症提供新的药物靶点。方法:提取PDK1基因条件性敲除型小鼠(PDK1-cKO)及野生型C57小鼠全骨髓细胞并诱导分化为破骨细胞,将诱导的破骨细胞进行抗酒石酸酸性磷酸酶染色,观察两组破骨细胞的数量及形态变化,蛋白免疫印迹法检测PDK1基因敲除后调控破骨细胞分化相关蛋白的表达。构建两组小鼠卵巢切除模型,采用Micro-CT扫描、抗酒石酸酸性磷酸酶染色来观察PDK1基因敲除对骨质疏松症的影响。结果与结论:①抗酒石酸酸性磷酸酶染色结果:相比较于野生型组,PDK1-cKO组破骨细胞数量在第4,6天显著减少(P<0.05);②蛋白免疫印迹结果显示:PDK1-cKO组调控破骨细胞分化的关键蛋白AKT磷酸化水平下降(P<0.05);③Micro-CT扫描结果:PDK1-cKO组比野生型组骨质疏松情况明显减轻(P<0.05);④抗酒石酸酸性磷酸酶染色结果:PDK1-cKO组胫骨远端破骨细胞较野生型组明显减少(P<0.05);⑤结果显示:PDK1基因可通过调控破骨细胞分化、优化骨代谢平衡来改善骨质疏松症状,有作为治疗骨质疏松疾病药物靶点的可能。展开更多
背景:目前CRISPR/Cas9结合Cre-loxP技术制备溴结构域蛋白4(bromodomain-containing protein 4,BRD4)基因敲除小鼠的实验方法非常少见。目的:运用CRISPR/Cas9技术敲除小鼠基因组中BRD4基因片段,构建BRD4基因敲除小鼠。方法:根据BRD4基因...背景:目前CRISPR/Cas9结合Cre-loxP技术制备溴结构域蛋白4(bromodomain-containing protein 4,BRD4)基因敲除小鼠的实验方法非常少见。目的:运用CRISPR/Cas9技术敲除小鼠基因组中BRD4基因片段,构建BRD4基因敲除小鼠。方法:根据BRD4基因的外显子序列,设计一段gRNA并合成。gRNA体外转录后和Cas9 mRNA以及含loxp位点的质粒共同注射入受精卵细胞中,Cas9通过识别gRNA先导链切割目的片段,然后loxp插入到切割位点,在与Cre配种后,Cre酶会切割loxp位点实现最终的特异性删除效果。注射后的受精卵细胞移植至C57BL/6N雌性小鼠获得子代小鼠,对子代小鼠进行测序鉴定其基因型。将成功导入loxp位点的小鼠BRD4-loxP^(+/-)(F0代)与野生型C57BL/6N小鼠配繁后筛选可稳定遗传的小鼠BRD4-loxP^(+/-)(F1代),BRD4-loxP^(+/-)小鼠一部分互相交配,一部分与CAGGCre-ER^(TM)鼠交配,获得BRD4-loxP^(+/+)小鼠和BRD4-loxP^(+/-)、CAGGCre-ER^(TM)共表达的小鼠(F2代);F2代小鼠互相交配可以获得纯合子BRD4-loxP^(+/+)、CAGGCre-ER^(TM)小鼠。将6-8周纯合子小鼠腹腔注射它莫昔芬75 mg/kg(溶于玉米油中),连续7 d即可完成BRD4基因的诱导敲除。取基因敲除后的小鼠尾部片段,吸附柱法提取DNA,通过琼脂糖凝胶电泳检测BRD4基因片段在小鼠尾部组织中的表达。结果与结论:①通过PCR筛选鉴定出F1代小鼠7,8,9,10,12,14,16,19,20,21,25,42,43和47号小鼠为成功插入2条loxP片段等位基因的杂合型小鼠;②F2代交互繁育出的F3代纯合子用它莫昔芬诱导后,经PCR凝胶电泳验证表明纯合子小鼠中BRD4基因片段被成功敲除;③提示利用CRISPR/Cas9技术结合Cre-loxP技术成功构建出了BRD4基因敲除小鼠。展开更多
Biological rhythms controlled by the circadian clock are absent in embryonic stem cells （ESCs）. However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biologic...Biological rhythms controlled by the circadian clock are absent in embryonic stem cells （ESCs）. However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells （iPSCs）. These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any dif- ference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before （DeBruyne et ah, 2007b）. After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.展开更多
文摘Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.
基金the Natural Science Foundation of Shandong Province,No. Y2008C54
文摘BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.
文摘Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.
基金supported in part by the National Institutes of Health through MD Anderson's Cancer Center Support Grant CA016672
文摘Mouse models of cancer enable researchers to learn about tumor biology in complicated and dynamic physiological systems. Since the development of gene targeting in mice, cancer biologists have been among the most frequent users of transgenic mouse models, which have dramatically increased knowledge about how cancers form and grow. The Chinese Journal of Cancer will publish a series of papers reporting the use of mouse models in studying genetic events in cancer cases. This editorial is an overview of the development and applications of mouse models of cancer and directs the reader to upcoming papers describing the use of these models to be published in coming issues, beginning with three articles in the current issue.
基金supported by the National Key Research and Development Plan of China(2017YFD0501602)the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five Plan(IDHT20170516)
文摘The classic method for gene knockout (KO) is based on homologous recombination (HR) and embryonic stem cell technique (Gerlai,1996).Actually,the procedure of homologous replacement is complicated and time consuming,although it has been popular during the past decades.Recent years,genome editing which can cause DNA sequence-specific mutations in the genomes of cellular
文摘背景:对于3-磷酸肌醇依赖性蛋白激酶1(3-phosphoinositide-dependent protein kinase 1,PDK1)的学术研究,国内外主要集中在内分泌和肿瘤学等学科领域,而在骨科学中关于调控破骨细胞对骨质疏松的改善尚未有系统研究与报道。目的:探索PDK1调控破骨细胞对骨质疏松症发病的影响及分子机制,为临床防治骨质疏症提供新的药物靶点。方法:提取PDK1基因条件性敲除型小鼠(PDK1-cKO)及野生型C57小鼠全骨髓细胞并诱导分化为破骨细胞,将诱导的破骨细胞进行抗酒石酸酸性磷酸酶染色,观察两组破骨细胞的数量及形态变化,蛋白免疫印迹法检测PDK1基因敲除后调控破骨细胞分化相关蛋白的表达。构建两组小鼠卵巢切除模型,采用Micro-CT扫描、抗酒石酸酸性磷酸酶染色来观察PDK1基因敲除对骨质疏松症的影响。结果与结论:①抗酒石酸酸性磷酸酶染色结果:相比较于野生型组,PDK1-cKO组破骨细胞数量在第4,6天显著减少(P<0.05);②蛋白免疫印迹结果显示:PDK1-cKO组调控破骨细胞分化的关键蛋白AKT磷酸化水平下降(P<0.05);③Micro-CT扫描结果:PDK1-cKO组比野生型组骨质疏松情况明显减轻(P<0.05);④抗酒石酸酸性磷酸酶染色结果:PDK1-cKO组胫骨远端破骨细胞较野生型组明显减少(P<0.05);⑤结果显示:PDK1基因可通过调控破骨细胞分化、优化骨代谢平衡来改善骨质疏松症状,有作为治疗骨质疏松疾病药物靶点的可能。
文摘Biological rhythms controlled by the circadian clock are absent in embryonic stem cells （ESCs）. However, they start to develop during the differentiation of pluripotent ESCs to downstream cells. Conversely, biological rhythms in adult somatic cells disappear when they are reprogrammed into induced pluripotent stem cells （iPSCs）. These studies indicated that the development of biological rhythms in ESCs might be closely associated with the maintenance and differentiation of ESCs. The core circadian gene Clock is essential for regulation of biological rhythms. Its role in the development of biological rhythms of ESCs is totally unknown. Here, we used CRISPR/CAS9-mediated genetic editing techniques, to completely knock out the Clock expression in mouse ESCs. By AP, teratoma formation, quantitative real-time PCR and Immunofluorescent staining, we did not find any dif- ference between Clock knockout mESCs and wild type mESCs in morphology and pluripotent capability under the pluripotent state. In brief, these data indicated Clock did not influence the maintaining of pluripotent state. However, they exhibited decreased proliferation and increased apoptosis. Furthermore, the biological rhythms failed to develop in Clock knockout mESCs after spontaneous differentiation, which indicated that there was no compensational factor in most peripheral tissues as described in mice models before （DeBruyne et ah, 2007b）. After spontaneous differentiation, loss of CLOCK protein due to Clock gene silencing induced spontaneous differentiation of mESCs, indicating an exit from the pluripotent state, or its differentiating ability. Our findings indicate that the core circadian gene Clock may be essential during normal mESCs differentiation by regulating mESCs proliferation, apoptosis and activity.
