Ple174-EGFP (Negative)

Ple174-EGFP Description:

The Ple174 promoter construct is derived from the promoter region of the receptor (calcitonin) activity modifying protein 3 (RAMP3) gene. The Pleiades Promoter Project created a 3439 bp MiniPromoter derived from a subsection of the RAMP3 promoter along with two downstream putative regulatory elements introduced it into the multiple cloning site (MCS) of Pleiades expression vector pEMS1306 (EGFP reporter flanked by minimal frt sites) to make plasmid pEMS1413 with Ple174 driving EGFP. The Ple174-EGFP (pEMS1413) 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) mEMS1204.03A. Four ESC lines (mEMS2970, mEMS3601, mEMS3603, and mEMS3627) were PCR validated. Chimeras were generated using mEMS2970.

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

Ple174-EGFP Expression Pattern:

No EGFP expression was detected above background in native fluorescence under epi or confocal microscopy. Anti GFP immunostaining (rabbit polyclonal, Invitrogen) did not detect specific GFP expression above background across numerous brain regions (brightfield).

Ple174 MiniPromoter Resources:

Ple174-EGFP
	plasmid pEMS1413 (in backbone pEMS1306)
	ESCs mEMS2970, mEMS3601, mEMS3603, mEMS3627
	mice generated using ESC mEMS2970
Resource Files
	Ple174 pEMS1413 Sequence	Jones lab
	Ple174 pEMS1413 MiniPromoter Design	Wasserman lab
	Ple174 pEMS1413 Vector NTI File  (Requires VectorNTI Sofware)	Holt lab
	Ple174 pEMS1413 Vector Map	Holt lab
	Ple174 pEMS1413 Genotyping Assay  (Requires VectorNTI Sofware)	Holt lab

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