Gene Targeting Cassettes

This page displays descriptions of the various gene targeting cassettes that are used in our 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 (25ug/ml). No restrictions on E. Coli host strain. Also weakly Kan resistant(20ug/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 (25ug/ml). Kan resistant(100ug/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 (25ug/ml). Kan resistant(100ug/ml) from bacterial EM7 promoter upstream of neo. 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 (25ug/ml). Kan resistant(100ug/ml) from bacterial EM7 promoter upstream of neo. 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 (25ug/ml). Kan resistant(100ug/ml) from bacterial EM7 promoter upstream of neo. 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(25ug/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(25ug/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(25ug/ml). No restrictions on E.Coli host strain.

Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DA. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat Biotechnol. 2004 May;22(5):589-94.
Skarnes WC, Moss JE, Hurtley SM, Beddington RS. Capturing genes encoding membrane and secreted proteins important for mouse development. Proc Natl Acad Sci USA. 1995 Jul 3;92(14):6592-6.