The Pleiades Promoter Project

All MiniPromoters

Ple111-EGFP (Positive)

Other constructs for this MiniPromoter: NONE

Other constructs for this Gene: Ple112-EGFP (Positive), Ple112-lacZ (Positive)


8.1 weeks, N2, Male (entire series)	8.1 weeks, N2, male (entire series)	Adult, N2, Male (entire series)

Ple111-EGFP Description:

The Ple111 promoter construct is derived from the promoter region of the Hypocretin (HCRT) gene. The Pleiades Promoter Project created a 544 bp MiniPromoter derived from a basal promoter element and introduced it into the multiple cloning site (MCS) of Pleiades expression vector pEMS1306 (EGFP reporter flanked by minimal frt sites) to make plasmid pEMS1417 with Ple111 driving EGFP. The Ple111-EGFP (pEMS1417) was introduced by homologous recombination as a single copy insertion into the Hprt1b-m3 locus in the X chromosome of mouse embryonic stem cells (ESCs) mEMS1204. Five ESC lines (mEMS1767, mEMS1768, mEMS1774, and mEMS1775) were PCR validated. Chimeras were generated using mEMS1774.

Note: Promoterless negative controls (pEMS1302, pEMS1306, pEMS1313) have been generated, as well as CAG positive controls (pEMS1157, pEMS1277 & pEMS1488).

Ple111-EGFP Expression Pattern:

A small cluster of EGFP-positive neurons is found in the hypothalamus. EGFP-immunoreactivity fills the cytoplasm of these cells, highlighting the cell body as well as extensive dendritic processes. This label is very specific to the hypothalamus and not seen in any other cell populations in the brain. Double label immunocytochemistry shows that a majority of these EGFP-positive hypothalamic cells co-express with the HCRT (orexin) neuropeptide (the gene from which this MiniPromoter was designed).

Ple111 MiniPromoter Resources:

Ple111-EGFP
	plasmid pEMS1417 (in backbone pEMS1306)
	ESCs mEMS1767, mEMS1768, mEMS1774, mEMS1775
	mice generated using ESC mEMS1774
	The mouse strain is available through the Jackson Laboratory: http://jaxmice.jax.org/strain/009115.html
Resource Files
	Ple111 pEMS1417 Sequence	Jones lab
	Ple111 pEMS1417 MiniPromoter Design	Wasserman lab
	Ple111 pEMS1417 Vector NTI File	Holt lab
	Ple111 pEMS1417 Vector Map	Holt lab
	Ple111 pEMS1417 Vector Map (Requires VectorNTI Sofware)	Holt lab
	Ple111 pEMS1417 Genotyping Assay	Simpson lab
	Ple111 pEMS1417 ESC description	Simpson lab
	Ple111 pEMS1417 Mouse description	Simpson lab

Publications:

Reference	PubMed ID
Shen HM, Nakamura A, Sugimoto J, Sakumoto N, Oda T, Jinno Y, Okazaki Y. Tissue specificity of methylation and expression of human genes coding for neuropeptides and their receptors, and of a human endogenous retrovirus K family. J Hum Genet. 2006;51(5):440-50. Epub 2006 Mar 17.	16544050
Waleh NS, Apte-Deshpande A, Terao A, Ding J, Kilduff TS. Modulation of the promoter region of prepro-hypocretin by alpha-interferon. Gene. 2001 Jan 10;262(1-2):123-8.	11179675

Sakurai T, Moriguchi T, Furuya K, Kajiwara N, Nakamura T, Yanagisawa M, Goto K. Structure and function of human prepro-orexin gene. J Biol Chem. 1999 Jun 18;274(25):17771-6.	10364220
Moriguchi T, Sakurai T, Takahashi S, Goto K, Yamamoto M. The human prepro-orexin gene regulatory region that activates gene expression in the lateral region and represses it in the medial regions of the hypothalamus. J Biol Chem. 2002 May 10;277(19):16985-92. Epub 2002 Feb 19.	11854267
Zhang S, Lin L, Kaur S, Thankachan S, Blanco-Centurion C, Yanagisawa M, Mignot E, Shiromani PJ. The development of hypocretin (orexin) deficiency in hypocretin/ataxin-3 transgenic rats. Neuroscience. 2007 Aug 10;148(1):34-43. Epub 2007 Jul 6.	17618058