Foxo1突变体转基因,ob/ob小鼠建立与表型分析
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摘要
Foxo1是瘦素作用的负调控因子,实验室前期工作发现,其突变体Foxo1ΔDBD可导致转基因小鼠体重降低等表现型,本实验旨在研究转基因小鼠此表型与瘦素作用的关系.首先采用ALZET?渗透泵的方法对成年雄性的具有瘦素基因缺陷(ob/ob)小鼠进行10 d的瘦素处理,使其获得生育能力,然后采用动物杂交的方法制备双基因改造鼠,即ob/ob基因遗传背景的Foxo1ΔDBD转基因小鼠(Foxo1ΔDBD/+,ob/ob).经PCR扩增的方法进行基因型鉴定后,对双基因改造鼠的体重、体温等表型进行分析.结果显示:①植入含瘦素溶液的ALZET?渗透泵10 d后的4只雄性ob/ob小鼠全部获得了生育能力.②成功制备了46只双基因改造鼠,39只ob/ob小鼠等.③表型分析结果显示,无ob/ob遗传背景的转基因Foxo1ΔDBD小鼠(Foxo1ΔDBD/+)与野生型小鼠相比,体重显著性降低(P<0.05),棕色脂肪内的UCP1转录水平显著升高(P<0.05);而双基因改造鼠和ob/ob小鼠相比,体重、体温、空腹血糖和采食量均无显著性差异(P>0.05),且UCP1转录水平上的表达也无显著性差异(P>0.05).本实验成功制备了双基因改造鼠,并进行了表型分析,结果表明Foxo1ΔDBD转基因小鼠的体重降低和棕色脂肪UCP1转录升高等表现依赖瘦素的作用.
Foxo1 was a negative regulator of leptin. In the preliminary work of the laboratory,its mutant Foxo1ΔDBD could cause phenotypes such as weight loss in transgenic mice. The purpose of this study was to investigate the relationship between this phenotype and leptin in transgenic mice. Firstly,the adult male leptin gene-deficient(ob/ob)mice were treated with leptin for 10 days to obtain fertility,and then the animal-hybridization method was used to prepare the double-gene modified mice. We found Foxo1ΔDBD transgenic mouse(Foxo1ΔDBD/+,ob/ob)with a genetic background of ob/ob gene. After genotyping by PCR amplification method,the phenotypes such as body weight and body temperature of the double gene-modified mice were analyzed. The results showed that:①Four male ob/ob mice after 10 days of implantation of the ALZET?osmotic pump containing leptin solution were all obtained fertility. ②46 double-gene engineered mice and 39 ob/ob mice were successfully prepared. ③Phenotypic analysis showed that transgenic Foxo1ΔDBD mice(Foxo1ΔDBD/+)without ob/ob genetic background showed significant weight loss was compared with wild-type mice(P<0.05),and UCP1 transcription level in brown fat was significant elevated(P<0.05). There was no significant difference in body weight,body temperature,fasting blood glucose and feed intake between the double-gene engineered and ob/ob mice(P>0.05),and the UCP1 transcription level was not significantly different in expression(P>0.05). In this experiment,double-gene engineered mice were successfully prepared and analyzed for phenotype. The results showed that the weight loss of Foxo1ΔDBD transgenic mice and the increaseofbrownfatUCP1 transcription level were dependent on leptin.
Foxo1 was a negative regulator of leptin. In the preliminary work of the laboratory,its mutant Foxo1ΔDBD could cause phenotypes such as weight loss in transgenic mice. The purpose of this study was to investigate the relationship between this phenotype and leptin in transgenic mice. Firstly,the adult male leptin gene-deficient(ob/ob)mice were treated with leptin for 10 days to obtain fertility,and then the animal-hybridization method was used to prepare the double-gene modified mice. We found Foxo1ΔDBD transgenic mouse(Foxo1ΔDBD/+,ob/ob)with a genetic background of ob/ob gene. After genotyping by PCR amplification method,the phenotypes such as body weight and body temperature of the double gene-modified mice were analyzed. The results showed that:①Four male ob/ob mice after 10 days of implantation of the ALZET?osmotic pump containing leptin solution were all obtained fertility. ②46 double-gene engineered mice and 39 ob/ob mice were successfully prepared. ③Phenotypic analysis showed that transgenic Foxo1ΔDBD mice(Foxo1ΔDBD/+)without ob/ob genetic background showed significant weight loss was compared with wild-type mice(P<0.05),and UCP1 transcription level in brown fat was significant elevated(P<0.05). There was no significant difference in body weight,body temperature,fasting blood glucose and feed intake between the double-gene engineered and ob/ob mice(P>0.05),and the UCP1 transcription level was not significantly different in expression(P>0.05). In this experiment,double-gene engineered mice were successfully prepared and analyzed for phenotype. The results showed that the weight loss of Foxo1ΔDBD transgenic mice and the increaseofbrownfatUCP1 transcription level were dependent on leptin.
引文
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[10] ZHANG J,WRIGHT W,BERNLOHR D A,et al. Alterations of the classic pathway of complement in adipose tissue of obesity and insulin resistance[J]. American Journal of Physiology-Endocrinology and Metabolism,2007,292(5):1433-1440.
[11] KLINGENBERG M. UCP1-A sophisticated energy valve[J]. Biochimie,2017,134:19-27.
[12] MUNEKATA K,SAKAMOTO K. Forkhead transcription factor Foxo1 is essential for adipocyte differentiation[J]. In Vitro Cellular&Developmental Biology-Animal,2009,45(10):642-651.
[13] HIGUCHI M,DUSTING G J,PESHAVARIYA H,et al. Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and forkhead box O1 mediated upregulation of antioxidant enzymes[J]. Stem Cells and Development,2012,22(6):878-888.
[14] ORTEGA MOLINA A,EFEYAN A,LOPEZ GUADAMILLAS E,et al. Pten positively regulates brown adipose function,energy expenditure,and longevity[J]. Cell metabolism,2012,15(3):382-394.
[2] ZHANG Y Y,PROENCA R,MAFFEI M,et al. Positional cloning of the mouse obese gene and its human homologue[J]. Nature,1994,372(6505):425-432.
[3] CORREIA M L G,HAYNES W G. Lessons from leptin’s molecular biology:potential therapeutic actions of recombinant leptin and leptin-related compounds[J]. Mini Reviews in Medicinal Chemistry,2007,7(1):31-38.
[4] ENRIORI P J,EVANS A E,SINNAYAH P,et al. Leptin resistance and obesity[J]. Obesity,2006,14(S8):254-258.
[5] KAESTNER K H,KN?CHEL W,MARTíNEZ D E. Unified nomenclature for the winged helix/forkhead transcription factors[J].Genes&Development,2000,14(2):142-146.
[6] RAMASWAMY S,NAKAMURA N,SANSAL I,et al. A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR[J]. Cancer Cell,2002,2(1):81-91.
[7] YANG G,LIM C Y,LI C,et al. FoxO1 inhibits leptin regulation of pro-opiomelanocortin promoter activity by blocking STAT3interaction with specificity protein 1[J]. Journal of Biological Chemistry,2009,284(6):3719-3727.
[8]周国丽,李浩,路万平,等.突变体转基因小鼠的建立[J].中国畜牧兽医,2018,45(3):612-619.
[9] LORD G M,MATARESE G,HOWARD J K,et al. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression[J]. Nature,1998,394(6696):897-901.
[10] ZHANG J,WRIGHT W,BERNLOHR D A,et al. Alterations of the classic pathway of complement in adipose tissue of obesity and insulin resistance[J]. American Journal of Physiology-Endocrinology and Metabolism,2007,292(5):1433-1440.
[11] KLINGENBERG M. UCP1-A sophisticated energy valve[J]. Biochimie,2017,134:19-27.
[12] MUNEKATA K,SAKAMOTO K. Forkhead transcription factor Foxo1 is essential for adipocyte differentiation[J]. In Vitro Cellular&Developmental Biology-Animal,2009,45(10):642-651.
[13] HIGUCHI M,DUSTING G J,PESHAVARIYA H,et al. Differentiation of human adipose-derived stem cells into fat involves reactive oxygen species and forkhead box O1 mediated upregulation of antioxidant enzymes[J]. Stem Cells and Development,2012,22(6):878-888.
[14] ORTEGA MOLINA A,EFEYAN A,LOPEZ GUADAMILLAS E,et al. Pten positively regulates brown adipose function,energy expenditure,and longevity[J]. Cell metabolism,2012,15(3):382-394.