Gene deletions by ends-in targeting in Drosophila melanogaster - PubMed (original) (raw)

Gene deletions by ends-in targeting in Drosophila melanogaster

Heng B Xie et al. Genetics. 2004 Nov.

Abstract

Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.

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Figures

F<sc>igure</sc> 1.—

Figure 1.—

The scheme for producing deletions. (A) The donor P element carrying regions from the left and the right of the gene to be deleted. (B) FLP and I-_Sce_I excise the donor DNA from the chromosome and generate a double-strand break within its A region, stimulating recombination with the endogenous A region. (C) The expected structure of the allele produced by recombination between the donor DNA and the target locus. Then, in a second step, I-_Cre_I is used to produce a double-strand break to the left of the whs marker gene. (D) If the chromosomal break is repaired by recombination within region A, the whs marker will be lost and a wild-type allele is produced. (E) If repair occurs by recombination within region C, the result will be a deletion of region B.

F<sc>igure</sc> 2.—

Figure 2.—

Molecular analysis of Sir2 targeting. (A) The donor construct. Restriction sites and fragment sizes are indicated for the wild-type allele (B) and the expected targeted allele (C). Small arrows in C indicate the primers used for diagnostic PCR. Southern blot results are shown in D, with marker sizes indicated at the left. The region used to probe the blot is indicated in C. Plus indicates genomic DNA from the wild type; T, DNA from flies carrying a targeted allele; B, _Bam_HI; K, _Kpn_I.

F<sc>igure</sc> 3.—

Figure 3.—

The crossing schemes for I-_Cre_I-mediated reduction at (A) Sir2, (B) Sirt2, and (C) p53.

F<sc>igure</sc> 4.—

Figure 4.—

Molecular analysis of Sir2 reduction. Restriction sites are shown for (A) the deletion allele, (B) the wild-type allele, and (C) a targeted allele in which part of whs has been deleted by NHEJ. Small arrows indicate the locations of primers used for the diagnostic PCR shown in D. In D, M indicates molecular weight markers with sizes indicated at the left and specific lanes are numbered for reference in E and F. (E and F) Southern blotting of _Kpn_I-digested DNA from wild type (+) and from the reduction alleles represented in lanes 4, 9, and 11. E was probed with probe 2 (indicated in B) and then stripped and reprobed with probe 3 (shown in F). K, _Kpn_I; B, _Bam_HI.

F<sc>igure</sc> 5.—

Figure 5.—

Molecular analysis of Sirt2 targeting. (A) The donor _P_-element construct, with the location of the roo element indicated. Restriction sites and coordinates, in kilobases, are given for (B) the wild-type allele and (C) the expected targeted allele. In C the small arrows indicate the locations of primers used for diagnostic PCR. The region used as a probe for Southern blotting is indicated. (D) Southern blotting of _Eco_RI-digested genomic DNA. “+” indicates wild type; lanes 1–5 indicate targeted alleles. Lanes 1, 3, and 5 represent the expected event. R, _Eco_RI.

F<sc>igure</sc> 6.—

Figure 6.—

Molecular analysis of Sirt2 reduction. The expected structures of (A) the wild-type allele, (B) the deletion allele, and (C) an allele produced by NHEJ repair of the I-_Cre_I break are indicated. _Eco_RI sites and their coordinates, in kilobases, are indicated. Small arrows indicate locations of primers used for diagnostic PCR. The regions used as probes for Southern blotting are also indicated. (D) Diagnostic PCR. (E and F) Southern blot results of _Eco_RI-digested DNA for selected alleles probed with probe 1 (E) and stripped and reprobed with probe 2 (F). M, molecular weight markers; +, wild-type allele; T, targeted allele; a and b, the expected deletion alleles; c, NHEJ reduction allele.

F<sc>igure</sc> 7.—

Figure 7.—

Expected results of p53 targeting. (A) The donor P element. (B) The expected structure of the targeted allele. Restriction sites and coordinates, in kilobases, are given. Small arrows indicate sites of primers used for diagnostic PCR. The region used as a probe for Southern blotting (Figure 8) is shown. R, _Eco_RI; N, _Not_I. The _Eco_RI at the 0 position is a combination of three sites very close to each other.

F<sc>igure</sc> 8.—

Figure 8.—

Molecular analysis of p53 targeting. (A) The structure that was deduced for the targeted allele. (B) The wild-type allele. Restriction sites and coordinates, in kilobases, are indicated, along with the region used as a probe for Southern blotting. (C) Southern blot of genomic DNA digested with _Eco_RI or _Not_I. T, targeted allele; +, wild-type allele; R, _Eco_RI; N, _Not_I.

F<sc>igure</sc> 9.—

Figure 9.—

Molecular analysis of p53 reduction alleles. The structures of (A) targeted, (B) wild-type, and (C) deletion alleles are indicated. (D) Southern blot of _Eco_RI-digested genomic DNA probed with probe 1. T, targeted allele; 1–7, reduction alleles; M, molecular weight markers. (E) Selected alleles, corresponding to the lane numbers in D, were examined in a second Southern blot of _Eco_RI-digested genomic DNA using probe 1. The blot was then stripped and reprobed with probe 2 (shown in F).

F<sc>igure</sc> 10.—

Figure 10.—

Proposed mechanism for generation of the p53 targeted allele. See text for details.

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