Far‐upstream elements are dispensable for tissue‐specific proenkephalin expression using a Cre‐mediated knock‐in strategy

Abstract

Several cis-regulatory DNA elements are present in the 5¢ upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cremediated gene knock-in strategy to target reporter constructs to a 'safe haven' loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5¢ upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5¢ ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved farupstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.

Computational promoter analysis of mouse, rat and human antimicrobial peptide-coding genes

“…Since Improbizer identified only six motifs, we first considered the top six motifs produced by each of these systems. Among the top six motifs, DMB- reported three motifs (TATA, AP-2, AP-1) that may bind TFs known to control the penk promoter [26,27]. MEME reported one motif (TATA) and Improbiser two (NF-Y, TATA) motifs.…”

“…Since DMB and MEME can identify arbitrary number of motifs, we also compared the top 20 motifs generated by DMB and MEME. Seven DMB-derived motifs coincided with known TFBSs (TATA, NF-kappaB, AP-2, AP-1 NFI/CTF, NF-Y, MZF1, MIG1, MBP-1) [26,27] known to control penk promoter. MEME yielded only three known penk promoter motifs (TATA, NFI/CTF, AP-1).…”

Controlled and localized genetic manipulation in the brain

“…Transgene insertion can be targeted with Cre recombinase after knockin of loxP elements. This so-called 'recombination-medi-ated cassette exchange' allows the creation of new transgenic mice carrying single integrants at predefined loci [38] to aid both comparative analyses [39] and also induction strategies [40].…”

Isolation and Characterization of SATB2, a Novel AT-rich DNA Binding Protein Expressed in Development- and Cell-Specific Manner in the Rat Brain

“…Previous work using a rat model indicated that 2.7 kb 5¢ upstream region of the ENK gene is both necessary and sufficient to govern correct cell-specific expression of the gene [6]. Our recent studies using the cre-lox ''knock-in'' strategy also indicated that the proximal 1.4 kb of the mouse ENK gene is sufficient for organ-specificity but farther upstream DNA elements are required for development-and cell-specific expression of the ENK gene [7]. This 2.7 kb region of the rat, mouse and human ENK genes contains an AT-rich DNA sequence [8][9][10] and interfering with the binding of proteins to this AT-rich DNA element resulted in an altered reporter gene expression [1,11,12].…”