Ple162-EGFP/cre (Positive)*

Ple162-EGFP/cre Description:

This Ple162 promoter construct is derived from the promoter region of the Pituitary homeobox 3 (PITX3) gene. The Pleiades Promoter Project created a 3636 bp MiniPromoter derived from an intronic candidate regulatory region and a segment of the PITX3 promoter and introduced it into the multiple cloning site (MCS) of Pleiades expression vector pEMS1302 (EGFP/cre reporter flanked by minimal frt sites) to make plasmid pEMS1148 with Ple162 driving EGFP/cre. The Ple162-EGFP/cre (pEMS1148) 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) mEMS21. The ESC lines mESM942, mEMS951, and mESM954 were PCR validated and chimeras were generated using mEMS954

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

Ple162-EGFP/cre Expression Pattern:

Expression of the Ple162-EGFP/cre results in the rearrangement of the Gt(ROSA)26tm1Sor reporter locus, thus producing strong ßGal activity in a discrete neuronal-like cell population in the ventral midbrain. In the mature brain, ßGal-positive cells are found surrounding the fasciculus retroflexus. Double labeling for ßGal and tyrosine hydroxylase shows that the ßGal-positive cells are not dopaminergic cells, but do border the dorsal edge of the main dopaminergic population of the VTA. A developmental analysis showed clear ßGal labeling of ventral midbrain cells in embryonic day 11.5 (E11.5) and E15.5 embryos as well as postnatal day 0 (P) and P7 pups, in the regions from and through which the dopaminergic population arises and migrates, respectively. Active expression of the EGFP/cre reporter gene product, however, cannot be detected in these cells by EGFP immunostaining, indicating ßGal expression is an historical marker of expression of the EGFP/cre reporter. Additionally, a substantial non-CNS staining was detected in muscle tissue of the abdominal wall, limb buds and posterior paravertebral tissues in E11.5 whole embryos as expected for the PITX3 gene.

Ple162 MiniPromoter Resources:

Ple162-EGFP/cre
	plasmid pEMS1148 (in backbone pEMS1302)
	ESCs mEMS942, mEMS951, mEMS954
	mice generated using ESC mEMS954
	The mouse strain is available through the Jackson Laboratory: http://jaxmice.jax.org/strain/008710.html
Resource Files
	Ple162 pEMS1148 Sequence	Jones lab
	Ple162 pEMS1148 MiniPromoter Design	Wasserman lab
	Ple162 pEMS1148 Vector NTI File  (Requires VectorNTI Sofware)	Holt lab
	Ple162 pEMS1148 Vector Map	Holt lab
	Ple162 pEMS1148 Genotyping Assay	Simpson lab

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