Gene Targeting Cassettes

This page displays descriptions of the various gene targeting cassettes that are used in the EUCOMM pipeline.

Promoter-Containing Targeting Cassettes

L1L2_Bact_P

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter	Reading frame-independent LacZ gene trap cassette: En2 splice acceptor / ECMV IRES / LacZ / SV40 polyadenylation site
Selectable Marker	Human β-actin promoter driving the wild-type neoR gene: human β-actin promoter / neomycin phosphotransferase (neoR) gene / SV40 polyA
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E. Coli host strain. Also weakly Kan resistant (20µg/ml), presumably from a cryptic bacterial promoter.

L1L2_PGK_P

The targeting cassette is flanked by Gateway L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter	Reading frame-independent LacZ gene trap cassette: En2 splice acceptor / ECMV IRES / LacZ / SV40 polyadenylation site
Selectable Marker	Phosphoglycerate Kinase 1 (PGK1) promoter driving the wild-type neoR gene: PGK promoter / neomycin phosphotransferase (neoR) gene / SV40 polyA
Plasmid	High copy number, ClonNAT resistant (25µg/ml). Kan resistant (100µg/ml) from bacterial EM7 promoter upstream of neo. No restrictions on E. Coli host strain.

Promoterless Targeting Cassettes

L1L2_gt0

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless, (reading frame 0) gene trap cassette. LacZ reporter gene and WT neomycin phosphotranferase (neoR) gene are translated from T2A oligopeptides1 as discreet proteins, not as fusions: En-2 splice acceptor (frame 0) / T2A / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E. Coli host strain.

L1L2_gt1

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless, (reading frame 1) gene trap cassette. LacZ reporter gene and WT neomycin phosphotranferase (neoR) gene are translated from T2A oligopeptides1 as discreet proteins, not as fusions: En-2 splice acceptor (frame 1) / T2A / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E. Coli host strain.

L1L2_gt2

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless, (reading frame 2) gene trap cassette. LacZ reporter gene and WT neomycin phosphotranferase (neoR) gene are translated from T2A oligopeptides1 as discreet proteins, not as fusions: En-2 splice acceptor (frame 2) / T2A / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E. Coli host strain.

L1L2_gtK

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker

Promoterless gene trap cassette containing an initiating ATG and optimal Kozak translation sequence. To be used as first coding exon downstream of untranslated exons lacking an initiating ATG. LacZ reporter gene and WT neomycin phosphotranferase (neoR) gene are translated from T2A oligopeptides1 as discreet proteins, not as fusions: En-2 splice acceptor (Kozak/ATG)/ T2A / LacZ / T2A / neoR / SV40 polyadenylation site

Plasmid

High copy number, ClonNAT resistant (25µg/ml). No restrictions on E. Coli host strain.

L1L2_st0

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless (reading frame 0) secretory trap cassette2 containing rat CD4 transmembrane (Tm) domain fused to N terminus of a lacZ reporter. WT neomycin phosphotranferase (neoR) gene is translated from a T2A oligopeptide1 as a discreet protein, not as a fusion protein: En-2 splice acceptor (frame 0) / ratCD4 Tm domain / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E.Coli host strain.

L1L2_st1

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless (reading frame 1) secretory trap cassette2 containing rat CD4 transmembrane (Tm) domain fused to N terminus of a lacZ reporter. WT neomycin phosphotranferase (neoR) gene is translated from a T2A oligopeptide1 as a discreet protein, not as a fusion protein: En-2 splice acceptor (frame 1) / ratCD4 Tm domain / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E.Coli host strain.

L1L2_st2

The targeting cassette is flanked by L1/L2 Gateway sites for insertion into R1/R2 Gateway sites upstream of the critical exon(s) in the intermediate vector.

Reporter / Selectable Marker	Promoterless (reading frame 2) secretory trap cassette2 containing rat CD4 transmembrane (Tm) domain fused to N terminus of a lacZ reporter. WT neomycin phosphotranferase (neoR) gene is translated from a T2A oligopeptide1 as a discreet protein, not as a fusion protein: En-2 splice acceptor (frame 2) / ratCD4 Tm domain / LacZ / T2A / neoR / SV40 polyadenylation site
Plasmid	High copy number, ClonNAT resistant (25µg/ml). No restrictions on E.Coli host strain.

¹Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA. (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving‘ 2A peptide-based retroviral vector. Nat Biotechnol. 22(5):589-94.

²Skarnes WC, Moss JE, Hurtley SM, Beddington RS. (1995) Capturing genes encoding membrane and secreted proteins important for mouse development. Proc Natl Acad Sci USA. 92(14):6592-6.