Generation of Mouse Embryonic Stem Cell Clones for Ghrelin Receptor Gene Knock Out_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WANG Weigang ¹ , WANG Zhigang ¹ , ZHANG Yongyan ² , FEI Jian ² , ZHENG Qi ¹

1.Department of General Surgery, The 6th Affiliated People’s Hospital of Shanghai Jiaotong University, Shanghai 200233, China;;
2.Shanghai Research Center for Biomodel Organism, Shanghai 201210, ChinaCorresponding Author: ZHENG Qi, E-mail: zhengqi1957@yahoo.com.cn;

Corresponding author：

Keywords：

Gene knock out; Embryonic stem cell; Ghrelin receptor

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Objective 　To establish the heterologous recombinant embryonic stem cell （ES cell） with ghrelin receptor (GHS-R) gene deletion in order to study the function of the GHS-R gene.
Methods 　PGK-neo cassette was replaced by TK-neo in X-pPNT agent. The target region was located in exon 1 and exon 2 of GHS-R. Two homologous arms were amplified from mouse ES cells genomic DNA and constructed into X-pPNT with SalⅠ/NotⅠand EcoRⅠ/BamHⅠ, respectively. ES cells were electrotransfected with the linearized targeting vector and screened with G418 and Gancyclovir. Finally, the positive ES cell clones were identified by PCR and sequencing.
Results 　The X-pPNT-TK-neo vector was obtained. And two homologous arms were inserted correctly. Finally, 328 positive clones were obtained by G418 and Gancyclovir screening, and 3 clones were confirmed as GHS-R gene homologous recombination.
Conclusion 　This study provides the necessary basis for the establishment of the GHS-R knock out mouse model and the further study on GHS-R gene function in vivo.

Citation： WANG Weigang,WANG Zhigang,ZHANG Yongyan,FEI Jian,ZHENG Qi. Generation of Mouse Embryonic Stem Cell Clones for Ghrelin Receptor Gene Knock Out. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(10): 733-738. doi: Copy

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，于运海, 等. 胃起搏对术后胃动力影响的实验研究 [Ｊ]. 中国普外基础与临床杂志, 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. 　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，于运海, 等. 胃起搏对术后胃动力影响的实验研究 [Ｊ]. 中国普外基础与临床杂志, 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Generation of Mouse Embryonic Stem Cell Clones for Ghrelin Receptor Gene Knock Out

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