4 Results
for term "sites"
- Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster...and often results in small insertion/deletion (indel) mutations at the site of the DSB (Lieber 2010). This causes frameshifting and the expression of mutated or nonfunctional protein. In contrast, homologous recombination (HR) uses a homologous template for repair to preserve genomic integrity. Gene ~~~
Figure 1Schematic outlining gene conversion using homology-directed repair from an integrated transgene. A donor plasmid consisting of a core cassette carrying the DNA of interest for incorporation (green) flanked by target homology arms (brown) is randomly inserted into the genome by P-element transposition. A genetic cross brings the target and donor together into the same cell. A DSB is induced in the target DNA to trigger HDR-mediated insertion of the DNA of interest into the target. A second genetic cross segregates the converted target and donor insertions. The final result (red dashed arrow) is insertion of donor sequence into the target site.
Figure 4HACK efficiencies across the second and third chromosome. (A–B) Conversion frequencies were tested for each donor and target pair on the (A) second or (B) third chromosome. The size of the circle represents the overall conversion rate of the target GAL4 line to all tested donor lines. Larger circles represent target GAL4 sites that were easier to convert (hot spots), while smaller circles represent target GAL4 sites that demonstrated low conversions (cold spots). For example, 18.2% of all F2 progeny of crosses from OK371-GAL4 and all seven second chromosome donors demonstrated conversions. The color indicates the relative conversion rate of the donor normalized to the highest conversion rate for the corresponding GAL4 lines. Thus colors indicate which donor line is most efficient for driving QF2G4H HACK conversions for a particular GAL4 line. For example, for OK371-GAL4, QF2G4H donor at 25C1 was the most effective (relative conversion rate 100%), and QF2G4H at 23F3 roughly two-thirds as effective (relative conversion rate 67%). Black and red arrows indicate the inserted transgene is in the forward and reverse orientation, respectively, relative to the reference Drosophila genome (Flybase R6.10).
Figure 6Donor and target inserted at the same loci do not necessarily lead to high conversion frequencies. (A) An attB-QF2G4H donor insert at the attP2 site is used to HACK Janelia GMR-GAL4 lines inserted at attP2. (B) Examples of QF2 lines that were HACKed from widely used attP2-GMR-GAL4 lines. Colabeling experiments demonstrate expression patterns of QF2G4H recapitulate those of the original attP2-GMR-GAL4 insertion. Bar, 100 µm (brains, left panel), 20 µm (inset magnification). (C) The conversion rate of attP2-GMR-GAL4 lines using attP2-QF2G4H or nearby QF2G4H lines. The attP2-QF2G4H donors were either in forward (black arrow, as generated by pBPGUw-HACK_G4 > QF2) or reverse (red arrow, as generated by pHACK_G4 > QF2) orientations. Both exhibited significantly higher success rates than three nearby donors (ANOVA, P < 0.001). The conversion rate mediated by attP2-QF2G4H in the same orientation as attP2-GMR-GAL4 was significantly higher than the conversion rate mediated by attP2-QF2G4H inserted in the opposite orientation (P = 4.7 × 10−5, Student’s t-test).