• 1.上海交通大學(xué)附屬第六人民醫(yī)院普外科(上海200233);;
  • 2.上海南方模式生物研究中心(上海201210);

目的 　建立生長激素促分泌素受體(ghrelin receptor，GHS-R)基因敲除小鼠胚胎干細(xì)胞（ES細(xì)胞）雜合子模型，為研究GHS-R基因的功能奠定基礎(chǔ)。
方法 　用TK-neo置換原X-pPNT載體的PGK-neo構(gòu)建目標(biāo)載體。以小鼠基因組DNA為模板, 用PCR方法擴(kuò)增2條同源臂，將其按照一定方向裝入含有TK-neo的X-pPNT載體，并測序鑒定。載體線性化及純化后電穿孔轉(zhuǎn)染小鼠ES細(xì)胞，用G418和更昔洛韋(Gancyclovir)對電穿孔轉(zhuǎn)染后的ES細(xì)胞進(jìn)行正、負(fù)篩選培養(yǎng)，得到雙藥抗性ES細(xì)胞，克隆后抽提基因組DNA，分別用PCR方法鑒定2條同源臂，并測序確定成功同源重組的ES細(xì)胞克隆。
結(jié)果 　改建X-pPNT載體成功，PCR獲得2條同源臂片段，測序正確，并按一定方向裝入打靶載體，ES細(xì)胞轉(zhuǎn)染后經(jīng)雙藥篩選得到328個(gè)陽性ES細(xì)胞克隆，PCR及測序鑒定證實(shí)3個(gè)克隆發(fā)生同源重組。
結(jié)論 　本研究成功獲得了GHS-R(－/+)雜合子小鼠ES細(xì)胞克隆，為進(jìn)一步通過顯微注射及雜交育種獲得GHS-R基因敲除小鼠打下了基礎(chǔ)。

引用本文： 王維剛,王志剛,張永彥,費(fèi)儉,鄭起. 生長激素促分泌素受體基因敲除小鼠胚胎干細(xì)胞的建立. 中國普外基礎(chǔ)與臨床雜志, 2008, 15(10): 733-738. doi: 復(fù)制

1. 　Howard AD, Feighner SD, Cully DF, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release [Ｊ]. Science, 1996; 273(5277)∶974.
2. 　Leung PK, Chow KB, Lau PN, et al. The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor [Ｊ]. Cell Signal, 2007; 19(5)∶1011.
3. 　Ghigo E, Broglio F, Arvat E, et al. Ghrelin： more than a natural GH secretagogue and/or an orexigenic factor [Ｊ]. Clin Endocrinol (Oxf), 2005; 62(1)∶1.
4. 　Dixit VD, Taub DD. Ghrelin and immunity: a young player in an old field [Ｊ]. Exp Gerontol, 2005; 40(11)∶900.
5. 　Locatelli V, Bresciani E, Bulgarelli I, et al. Ghrelin in gastroenteric pathophysiology [Ｊ]. J Endocrinol Invest, 2005; 28(9)∶843.
6. 　Kitazawa T, De Smet B, Verbeke K, et al. Gastric motor effects of peptide and non-peptide ghrelin agonists in mice in vivo and in vitro [Ｊ]. Gut, 2005; 54(8)∶1078.
7. 　Peeters TL. Ghrelin: a new player in the control of gastrointestinal functions [Ｊ]. Gut, 2005; 54(11)∶1638.
8. 　Fujino K, Inui A, Asakawa A, et al. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats [Ｊ]. J Physiol, 2003; 550(Pt 1)∶227.
9. 　Tack J, Depoortere I, Bisschops R, et al. Influence of ghrelin on interdigestive gastrointestinal motility in humans [Ｊ]. Gut, 2006; 55(3)∶327.
10. 　Dornonville de la Cour C, Lindstrm E, Norlén P, et al. Ghrelin stimulates gastric emptying but is without effect on acid secretion and gastric endocrine cells [Ｊ]. Regul Pept, 2004; 120(1-3)∶23.
11. 　Tschp M, Wawarta R, Riepl RL, et al. Post-prandial decrease of circulating human ghrelin levels [Ｊ]. J Endocrinol Invest, 2001; 24(6)∶RC19.
12. 　Tack J, Depoortere I, Bisschops R, et al. Influence of ghrelin on gastric emptying and meal-related symptoms in idiopathic gastroparesis [Ｊ]. Aliment Pharmacol Ther, 2005; 22(9)∶847.
13. 　李富宇， 何生， 于運(yùn)海, 等. 胃起搏對術(shù)后胃動(dòng)力影響的實(shí)驗(yàn)研究 [Ｊ]. 中國普外基礎(chǔ)與臨床雜志, 2003; 10(1)∶33.
14. 　Dembinski A, Warzecha Z, Ceranowicz P, et al. Ghrelin attenuates the development of acute pancreatitis in rat [Ｊ]. J Physiol Pharmacol, 2003; 54(4)∶561.
15. 　Cassoni P, Papotti M, Catapano F, et al. Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue [Ｊ]. J Endocrinol, 2000; 165(1)∶139.
16. 　Jeffery PL, Murray RE, Yeh AH, et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines [Ｊ]. Endocr Relat Cancer, 2005; 12(4)∶839.
17. 　Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding [Ｊ]. Nature, 2001; 409(6817)∶194.
18. 　Sun Y, Wang P, Zheng H, et al. Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor [Ｊ]. Proc Natl Acad Sci USA, 2004; 101(13)∶4679.
19. 　Sun Y, Butte NF, Garcia JM, et al. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance [Ｊ]. Endocrinology, 2008; 149(2)∶843.
20. 　Hasty P, Rivera-Pérez J, Chang C, et al. Target frequency and integration pattern for insertiond replacement vectors in embryonic stem cells [Ｊ]. Mol Cell Biol, 1991; 11(9)∶4509.
21. 　Deng C, Capecchi MR. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus [Ｊ]. Mol Cell Biol, 1992; 12(8)∶3365.
  1. 1. 　Howard AD, Feighner SD, Cully DF, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release [Ｊ]. Science, 1996; 273(5277)∶974.
  2. 2. 　Leung PK, Chow KB, Lau PN, et al. The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor [Ｊ]. Cell Signal, 2007; 19(5)∶1011.
  3. 3. 　Ghigo E, Broglio F, Arvat E, et al. Ghrelin： more than a natural GH secretagogue and/or an orexigenic factor [Ｊ]. Clin Endocrinol (Oxf), 2005; 62(1)∶1.
  4. 4. 　Dixit VD, Taub DD. Ghrelin and immunity: a young player in an old field [Ｊ]. Exp Gerontol, 2005; 40(11)∶900.
  5. 5. 　Locatelli V, Bresciani E, Bulgarelli I, et al. Ghrelin in gastroenteric pathophysiology [Ｊ]. J Endocrinol Invest, 2005; 28(9)∶843.
  6. 6. 　Kitazawa T, De Smet B, Verbeke K, et al. Gastric motor effects of peptide and non-peptide ghrelin agonists in mice in vivo and in vitro [Ｊ]. Gut, 2005; 54(8)∶1078.
  7. 7. 　Peeters TL. Ghrelin: a new player in the control of gastrointestinal functions [Ｊ]. Gut, 2005; 54(11)∶1638.
  8. 8. 　Fujino K, Inui A, Asakawa A, et al. Ghrelin induces fasted motor activity of the gastrointestinal tract in conscious fed rats [Ｊ]. J Physiol, 2003; 550(Pt 1)∶227.
  9. 9. 　Tack J, Depoortere I, Bisschops R, et al. Influence of ghrelin on interdigestive gastrointestinal motility in humans [Ｊ]. Gut, 2006; 55(3)∶327.
  10. 10. 　Dornonville de la Cour C, Lindstrm E, Norlén P, et al. Ghrelin stimulates gastric emptying but is without effect on acid secretion and gastric endocrine cells [Ｊ]. Regul Pept, 2004; 120(1-3)∶23.
  11. 11. 　Tschp M, Wawarta R, Riepl RL, et al. Post-prandial decrease of circulating human ghrelin levels [Ｊ]. J Endocrinol Invest, 2001; 24(6)∶RC19.
  12. 12. 　Tack J, Depoortere I, Bisschops R, et al. Influence of ghrelin on gastric emptying and meal-related symptoms in idiopathic gastroparesis [Ｊ]. Aliment Pharmacol Ther, 2005; 22(9)∶847.
  13. 13. 　李富宇， 何生， 于運(yùn)海, 等. 胃起搏對術(shù)后胃動(dòng)力影響的實(shí)驗(yàn)研究 [Ｊ]. 中國普外基礎(chǔ)與臨床雜志, 2003; 10(1)∶33.
  14. 14. 　Dembinski A, Warzecha Z, Ceranowicz P, et al. Ghrelin attenuates the development of acute pancreatitis in rat [Ｊ]. J Physiol Pharmacol, 2003; 54(4)∶561.
  15. 15. 　Cassoni P, Papotti M, Catapano F, et al. Specific binding sites for synthetic growth hormone secretagogues in non-tumoral and neoplastic human thyroid tissue [Ｊ]. J Endocrinol, 2000; 165(1)∶139.
  16. 16. 　Jeffery PL, Murray RE, Yeh AH, et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines [Ｊ]. Endocr Relat Cancer, 2005; 12(4)∶839.
  17. 17. 　Nakazato M, Murakami N, Date Y, et al. A role for ghrelin in the central regulation of feeding [Ｊ]. Nature, 2001; 409(6817)∶194.
  18. 18. 　Sun Y, Wang P, Zheng H, et al. Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor [Ｊ]. Proc Natl Acad Sci USA, 2004; 101(13)∶4679.
  19. 19. 　Sun Y, Butte NF, Garcia JM, et al. Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance [Ｊ]. Endocrinology, 2008; 149(2)∶843.
  20. 20. 　Hasty P, Rivera-Pérez J, Chang C, et al. Target frequency and integration pattern for insertiond replacement vectors in embryonic stem cells [Ｊ]. Mol Cell Biol, 1991; 11(9)∶4509.
  21. 21. 　Deng C, Capecchi MR. Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus [Ｊ]. Mol Cell Biol, 1992; 12(8)∶3365.