Thepresenceofbothnegative andpositive ele tisin the5'-flanking sequenceoftheratNa,K-ATPase oa3 subunit gene arerequired forbrain expression in transgenic mice

TheNa,K-ATPase isan integral plasmamembrane protein consisting ofcaand subunits, eachofwhich hasdiscrete isoforms expressed ina tissue-specific manner.Ofthethree functional a isoform genes,the one encoding thea3 isoform isthemosttssuerestricted inits expression, being foundprimarily Inthe brain. ToIdentify regions ofthea3 isoformgenethat areinvolved indirecting expression inthebrain, a1.6 kb5'-flanking sequencewas attached toa reporter gene,chloramphenicol acetyltransferase (CAT). The a3- CATchimeric geneconstruct was microlnjected into fertilized mouse eggs,andtransgenic micewere produced. Analysis ofadulttransgenic micefrom different lines revealed thatthetransgene isexpressed primarily inthebrain. Tofurther delineate regions that areneededforconferring expression inthistissue, systematic deletions ofthe5'-flanking sequenceofthe a3- CATfusion constructs weremadeandanalyzed, againusing transgenic mice.Theresults fromthese analyses indicate thatDNA sequencesrequired for mediating brain-specific expression ofthea3 Isoform gene are present within210bpupstream of the transcription initiation site.a3- CAT promoter constructs containing scanning mutations Inthisregion werealsoassayed intransgenic mice. Thesestudies haveidentified botha functional neural-restrictive silencer element as wellas a positively acting cis element.