Human gene targeting favors insertions over deletions - PubMed (original) (raw)
Human gene targeting favors insertions over deletions
David W Russell et al. Hum Gene Ther. 2008 Sep.
Abstract
Gene targeting is a powerful technique for manipulating the human genome, but few studies have directly compared the targeting frequencies of various types of vector constructs. Here we show that similar targeting constructs are able to insert nucleotides at the homologous chromosomal target locus more efficiently than they can delete nucleotides, and combination insertion/deletion vectors appear to target at intermediate frequencies. This holds true for deletions ranging from 1 to 334 bp and insertions ranging from 1 to 1332 bp. In addition, vectors designed to inactivate the human hypoxanthine phosphoribosyltransferase gene (HPRT) by deleting nucleotides often produced rearrangements at the target locus that in many cases were due to insertions of multimerized vector constructs, effectively converting a deletion vector into an insertion vector. These findings were obtained when adeno-associated virus vectors were used to efficiently deliver single-stranded DNA targeting constructs, but the same phenomenon was also observed when transfecting linearized double-stranded plasmids. Thus human cells distinguish between deletion and insertion vectors and process their recombination intermediates differently, presumably at the heteroduplex stage, with implications for the design of gene-targeting vectors and the evolution of human genomes.
Figures
FIG. 1.
Targeting HPRT with insertion and deletion vectors. (A) The structures of five AAV gene-targeting vectors and a section of the human chromosomal HPRT locus are shown with the viral inverted terminal repeats as hatched boxes, homology arms as gray boxes, and HPRT exons as black boxes; Δ, deletions. Locations of the PGK promoter; neo gene; _Hin_dIII (H), _Ahd_I (A), and _Bso_BI (B) restriction sites; and probe used in Southern blots are also shown. (B) The fraction of colonies resistant to 6TG (solid columns) or G418 (open columns) obtained after infection of normal human male fibroblasts (MHF2 cells) with the indicated AAV-HPe3-based vector is shown (mean ± standard deviation, n : 3). The hatched column represents the fraction of 6TG-resistant colonies obtained without infection (mean of six experiments in which 6TG-resistant colonies were obtained only once). (C) Southern blots of representative 6TG-resistant MHF2 clones transduced with AAV-HPe3PN, AAV-HPe3PN(Δ334), or AAV-HPe3(Δ334) as indicated. The left and right lanes represent parental, untransduced cells. Each lane contains genomic DNA digested with _Hin_dIII and _Ahd_I and probed as shown in (A). The positions of size standards are shown on the left. (D) As in (C) but done in HT-1080 cells.
FIG. 2.
Structures of HPRT loci targeted with AAV-HPe3(+4) and AAV-HPe3(Δ4). (A) Southern blots of representative 6TG-resistant fibroblast clones transduced with the AAV-HPe3(+4) or AAV-HPe3(Δ4) targeting vectors and portrayed as in Fig. 1C. Genomic DNA was digested with _Bso_BI and probed as in Fig. 1. (B–G) Possible recombination events that would produce targeting at the HPRT locus are shown with the AAV terminal repeats as hatched boxes, homology arms as gray boxes, HPRT exons as black boxes; B, _Bso_BI sites. Recombination crossovers are shown as crossed lines. Predicted sizes of fragments on the Southern blot are indicated.
FIG. 3.
Correction of AP mutations by gene targeting. (A) The structure of retroviral MLV-LAPIH-based vectors are shown with murine leukemia virus long terminal repeats (LTRs), AP gene, internal ribosome entry site (IRES), hygromycin resistance gene, and the +1, Δ1, and wild-type (WT) sequences surrounding the bp 375 mutation site indicated above the AP gene. The AAV-5′APBss targeting vector is shown with hatched boxes representing the inverted terminal repeats, homology to the MLV-LAPIH elements indicated, a 5′ portion of the wild-type AP gene, and the _Bss_HII sites used to prepare the targeting vector. (B) Gene-targeting frequencies of immortalized fibroblasts containing +1 (solid column) or Δ1 (open column) AP mutations and transduced with AAV-5′APBss are shown as the number of AP+ foci obtained per 105 infected cells (mean ± standard deviation, n = 3).
FIG. 4.
Correction of HPRT mutations with transfected plasmid constructs or AAV vectors. (A) Structures of the linear plasmid fragment used for targeting, the AAV-HPe3 targeting vector, and the HPRT target loci containing +4 or Δ4 mutations in exon 3 are shown with the AAV terminal repeats as hatched boxes, homology arms as gray boxes, and HPRT exons as black boxes. (B) The fraction of HAT-resistant colonies obtained from untransfected, untransduced cells, cells transduced with AAV-HPe3, and cells transfected with the pHPe2/3 plasmid fragment is shown, with each column representing one experimental value. Two clones (c1 and c2), containing each type of mutation as indicated, were tested. Asterisks indicate that no HAT-resistant colonies were obtained and the columns below the asterisks show the maximal possible value given the sensitivity of the experiment. The lower cell number used when infecting with AAV reduced the sensitivity of the experiment with the +4-c2 clone, and thus the true targeting frequency could be lower in this case where a deletion was being introduced.
References
- Bill C.A. Taghian D.G. Duran W.A. Nickoloff J.A. Repair bias of large loop mismatches during recombination in mammalian cells depends on loop length and structure. Mutat. Res. 2001;485:255–265. - PubMed
- Chamberlain J.R. Schwarze U. Wang P. Hirata R.K. Hankenson K.D. Pace J.M. Underwood R.A. Song K.M. Sussman M. Byers P.H. Russell D.W. Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science. 2004;303:1198–1201. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous