The effects of polymorphisms on human gene targeting

Abstract

DNA mismatches that occur between vector homology arms and chromosomal target sequences reduce gene targeting frequencies in several species; however, this has not been reported in human cells. Here we demonstrate that even a single mismatched base pair can significantly decrease human gene targeting frequencies. In addition, we show that homology arm polymorphisms can be used to direct allele-specific targeting or to improve unfavorable vector designs that introduce deletions.

Figure 1.

Gene targeting at MLV target loci with SNPs. (A) Schematic of the AAV-HSN5′ targeting vector and six MLV provirus loci with the location of the 53 bp neo gene deletion (Δ53) and the G to A SNPs shown. Targeting frequencies were measured as the fraction of G418-resistant colonies obtained after infecting polyclonal HT-1080 populations containing each indicated MLV provirus with AAV-HSN5′. LTR, long terminal repeat; hph, hygromycin phosphotransferase; S, SV40 promoter; T, Tn5 bacterial promoter, 5′neo, truncated neo gene; ori, p15A plasmid origin; *P < 0.05 versus LHSNΔ53O. (B) Graphic illustration of SNPs present in the recovered targeted loci. Colors indicate the presence of MLV target SNPs (red), AAV vector SNPs (blue) or segments between identifiable SNPs (gray).

Figure 2.

Targeting in trisomic iPSC cells containing three different SNP haplotypes. (A) APP locus showing AAV-APPe3ITKNA targeting vector, forward (F1) and reverse (R1, R2) PCR primers, SNP locations and sequences found in each of three APP alleles. (B) Representative sequence reads are shown demonstrating targeting at the GT allele in a Down syndrome iPSC clone. (C) The percentage of targeted APP alleles with each SNP haplotype.

Figure 3.

Gene targeting with a deletion vector. Illustration of MLV provirus targets with a 4-bp neo insertion (ins4) and different flanking deletions (Δ1, Δ4), with their targeting frequencies as shown in Figure 1. *P < 0.05 versus LHSNins4O.

Figure 4.

Correction of HPRT mutations with AAV and plasmid vectors. (A) Structure of the HPRT locus containing either a 4-bp deletion or insertion in exon 3, the AAV-HPe3 and AAV-Hpe3(i2i3+1) targeting vectors used and the analogous plasmid-based targeting constructs pHPe2/3 and pHPe2/3(i2i3+1). Gene targeting frequencies are shown as the fraction of HAT-resistant colonies obtained after transducing HT-1080 subclones harboring HPRT mutations with either AAV targeting vectors (*P < 0.05 versus AAV-HPe3) (B) or linearized plasmid targeting constructs (C).