5’-end arm ~ 3-5Kb long, usually doesn’t contain gene promoter
betaGal/neo/polyA – promoter less trapping cassette, located in intron after start of transcription, contains splice acceptor site (SA). In-frame betaGal and neo fusion expression is driven by targeted gene promoter, flanked by FRT sites. Trapping cassette must contain mammalian selection marker, 5’end LoxP, 2 flanking FRT sites and efficient polyA “trapping” sequence to terminate transcription.
critical exon(s) – essential for gene functionality, deletion disrupts gene function 2 ways: removing functional domain and creating a frame shift in gene expression
Lox71 – taccgttcgtatagcatacattatacgaagttat
LoxP – ataacttcgtatagcatacattatacgaagttat
Lox71 - LoxP genomic distance is ~1Kb, flank a critical exon, work as a pair for conditional cre mediated in vivo deletion of critical exon in mouse
FRT - gaagttcctattccgaagttcctattctctagaaagtataggaacttc
two FRT sites used for flp mediated in vivo removal of betaGal/neo-LoxP cassette in ES cells.
3’-end arm ~ 3-5Kb long
DTA - diphtheria toxin fragment A - PGK promoter driven mammalian negative
selection marker, replaces more conventional TK negative selection, requires FIAU or gancyclovirin.
Vector – remaining portion of the vector after linearization with AsiSI, protects DTA from nucleases after electroporation into ES cells