A chromosomal position effect on gene targeting in human cells

Abstract

We describe gene targeting experiments involving a human cell line (RAN10) containing, in addition to its endogenous alleles, two ectopic alleles of the interferon-inducible gene 6-16. The frequency of gene targeting at one of the ectopic 6-16 alleles (H3.7) was 34-fold greater than the combined frequency of gene targeting involving endogenous 6-16 alleles in RAN10. Preference for H3.7 was maintained when the target loci in RAN10 were transcriptionally activated by interferon. Despite the 34-fold preference for H3.7, the absolute gene targeting efficiency in RAN10 was only 3-fold higher than in the parental HT1080 cell line. These data suggest that different alleles can compete with each other, and perhaps with non-homologous loci, in a step which is necessary, but not normally rate-limiting, for gene targeting. The efficiency of this step can therefore be more sensitive to chromosomal position effects than the rate-determining steps for gene targeting. The nature of the position effects involved remains unknown but does not correlate with transcription status, which in our system has a very modest influence on the frequency of gene targeting. In summary, our work unequivocally identifies a position effect on gene targeting in human cells.

Figure 1

(A) The 6–16 gene targeting system. (a) Targeting construct p6gpt with vector sequences removed. (b) The 6–16 gene. (c) The product of gene targeting involving (a) and (b). (B) SalI-cut p6–16neo, used to generate ectopic 6–16 alleles. DNA is represented as follows: black boxes, 6–16 gene exons, numbered 1–5; white ellipse, IFN-responsive region of the 6–16 gene promoter; thick lines, other 6–16 gene DNA; stippled boxes, multiple cloning sites of pBSIIKS+; thin line, other pBluescript DNA; white boxes, drug resistance cassettes; hatched box, SV40 early promoter; black bar, target-specific probe in Southern analyses; short horizontal arrows, primers used in PCR assay for targeting. A, Asp718; S, SalI.

Figure 2

Structure of 6–16 alleles in RAN10. Genomic DNA was digested with HindIII (A), BglII (B) or BstZ17I and NotI (C), blotted and probed with the target-specific probe shown in Figure 1. Genomic DNA was from HT1080, RAN10 or targeted RAN10 clones (RAN10T1, etc.) whose MPA resistance was partially IFN-dependent. Size markers (M or λ) are described in Materials and Methods and sizes (kb) of arrowed markers are indicated. (D) Schematic representation of the endogenous (a) and ectopic copies (b and c) of 6–16 in RAN10. DNA is represented as in Figure 1. Relevant sites are shown for Asp718 (A), BglI (Bi), BglII (Bii), BstZ17I (Bs), HindIII (H), PvuII (Pv), MfeI (M), NotI (N) and SacI (S).

Figure 3

Evidence for constitutive 6–16 transcription of the H3.7 allele in RAN cell lines. (A) Northern blot of RNA isolated from cell lines grown with or without IFN treatment, as indicated. (B) Tests for IFN dependence of resistance to MPA. Cells were seeded at low density and grown in selective medium with or without IFN (Materials and Methods). Results for RAN10 and five p6gpt-transfected, MPA^r derivatives are shown. RAN10T4 was not targeted at a 6–16 allele, as judged by PCR and Southern analyses. The remaining clones were targeted at an endogenous allele (RAN10T4I and RAN10T46I) or at the H3.7 allele (RAN10T31 and RAN10T36), as judged by PCR and Southern analyses.

Figure 4

Southern blot analyses of HindIII-digested genomic DNA from HT1080, RAN10 and nine representative p6gpt-transfected RAN10 derivatives. In total, 108 MPA^r, PCR-positive clones, derived from experiments 4–6 (Table 1), were analysed in this way and each generated one of the 10 representative hybridisation patterns shown in lanes 2–11, as indicated below the autoradiograph. The probe was as shown in Figure 1A.

Figure 5

Three models to explain preferential targeting to H3.7 without increased ATF. Regions of homology are shown as white boxes. Hatched and stippled regions represent chromosomal flanking sequences at H3.7 and other 6–16 alleles, respectively. RLS, rate-limiting step. (A) Competition between H3.7 and other alleles for interaction with limiting amounts of targeting construct. (B) Competition between H3.7 and other 6–16 alleles for recruitment of a limiting factor (ellipse) required for HR. (C) Interallelic interference. (a) The H3.7 allele physically interacts with endogenous 6–16 alleles, preventing access by the targeting construct. (b) Endogenous 6–16 alleles do not impair access to the H3.7 allele by the targeting construct. See text for further details.