Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A ) Donor vectors for knock-in. pHDR-T2A-LexA-GAD-Hsp70-3xP3-RFP and pHDR-T2A-QF2-Hsp70-3xP3-RFP contain T2A-LexA-GAD or QF2 transcriptional activators followed by HSP70 terminators. pHDR-T2A-QF2-T2A-LexA-GAD-3xP3-RFP contains both activators flanked by FRT sites followed by SV40 terminator. All the vectors contain a 3XP3-RFP transformation marker flanked by loxP sites. ( B ) Long homology arm cloning method. ~1000 bp homology arms are amplified from genomic DNA and inserted into the AscI and SacI sites by Gibson assembly. A separate target-gene-specific guide RNA is cloned into U6 promoter expression vector such as pCFD3. ( C ) Drop-in cloning method. Based on the gRNA-int200 method previously described . A company synthesizes and clones a DNA fragment into the pUC57_Kan_gw_OK2 vector. The resulting plasmid contains the following elements: (1) two guide RNAs under the control of a U6 promoter, one (gRNA1) targeting the vector (pink arrows) to linearize the homology donor in vivo, and another (gRNA geneX ) targeting the gene of interest; (2) a tRNA sequence to allow liberation of the individual guides by the endogenous tRNA processing machinery; (3) 200 bp short homology arms; and (4) BbsI and SacI cloning sites. The AscI/SacI T2A-LexA-GAD, T2A-QF, or T2A-LexA-GAD-T2A-QF fragments can then be ligated in a single directional cloning step into the BbsI/SacI sites to produce the donor plasmid. ( D ) T2A-LexA-GAD and ( E ) T2A-QF2 knock-in strategy. CRISPR-based HDR causes integration of the T2A-LexA-GAD or T2A-QF2 in the most 5’ coding exon common to all or most isoforms, resulting in expression of the activators under control of the endogenous gene regulatory region. The knock-in also produces a truncated endogenous protein and thus a strong loss-of-function allele.

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Knock-In, Transformation Assay, Marker, Clone Assay, Amplification, Expressing, Plasmid Preparation, Control, In Vivo, Sequencing, CRISPR

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: T2A-LexA-GAD and T2A-QF2 knock-in lines.

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Knock-In

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A–KK ) T2A-LexA-GAD knock-in lines crossed to a LexAop-GFP reporter and T2A-QF2 knock-in lines crossed to a QUAS-GFP reporter. Panels show third instar larva. GFP shows the driver line expression pattern. RFP shows the 3XP3 transformation marker, which labels the posterior gut and anal pads of the larva. Gene names and tissues are on the left. We failed to obtain LexA-GAD knock-ins for Mef2 ( E ) and He (DD). (LL–MM) Third instar imaginal disc from the insertions in the nubbin (nub ) gene. Note that most of the lines are highly tissue-specific and are comparable between the LexA-GAD and QF2 knock-ins. Insertions in the daughterless gene ( da) and nub are an exception, as the T2A-LexA-GAD, but not the T2A-QF2, gives the expected expression pattern. Insertions in the gut-specific genes mex1 ( X–Y ) and Myo31Df ( Z–AA ) also differed between the LexA-GAD and QF2 drivers.

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Knock-In, Expressing, Transformation Assay, Marker

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A ) T2A-QF2-3XP3-RFP in the repo gene crossed to a QUAS-GFP reporter. In third instar larva, the reporter is expressed in the expected glial cells, but also misexpressed in gut and anal pad (yellow asterisk). ( B ) T2A-QF2 in the repo gene with the 3XP3-RFP removed by Cre-Lox recombination, crossed to a QUAS-GFP reporter. Removal of 3XP3-RFP eliminated gut and anal pad misexpression and did not affect glial cell expression (white arrowheads).

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Expressing

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A ) CRISPR-based HDR strategy for integration of the T2A-QF2-T2A-LexA-GAD-3XP3 in the most 5’ coding exon common to all or most isoforms, resulting in expression of both activators under control of the endogenous gene regulatory region. The knock-in also produces a truncated endogenous protein and thus a strong loss-of-function allele. If desired, one of the two coding regions can then be excised with Flp, resulting in flies that express only QF2 or LexA-GAD. ( B ) Alternative strategy allows gene enhancers to be cloned upstream of of T2A-QF2-T2A-LexA-GAD. The vector backbone includes an attB site for phiC31 insertion into attP flies. ( C–D ) T2A-QF2-T2A-LexA-GAD knock-ins crossed to a QUAS-GFP+LexAop-mCherry double reporter line. ( C ) The elav T2A-QF2-T2A-LexA-GAD knock-in drives both QUAS-GFP and LexAop-mCherry in the larval brain. There is some leakiness of mCherry in the body wall muscle (arrowheads). ( D ) The hh T2A-QF2-T2A-LexA-GAD knock-in drives both QUAS-GFP and LexAop-mCherry in the posterior of the wing imaginal disc. GFP expression is much less than mCherry in the wing pouch (asterisks). ( E–F ) Enhancer-T2A-QF2-T2A-LexA-GAD lines crossed to a QUAS-GFP+LexAop-mCherry double reporter line. ( E ) The dpp-blk enhancer-T2A-QF2-T2A-LexA-GAD line drives both QUAS-GFP and LexAop-mCherry along the anterior/posterior boundary of the wing imaginal disc. GFP expression is much less than mCherry in the wing pouch (stars). ( F ) The Ilp2 enhancer-T2A-QF2-T2A-LexA-GAD line drives both QUAS-GFP and LexAop-mCherry in the insulin-producing cells of the larval brain (arrows). The fat body mCherry expression (yellow arrowhead) is from leakiness of the reporter stock and does not indicate LexA-GAD activity.

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: CRISPR, Expressing, Control, Knock-In, Clone Assay, Plasmid Preparation, Activity Assay

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A ) FLP/FRT recombination scheme. Flies containing both hs-FLP and a T2A-QF2-T2A-LexA-GAD knock-in are heat shocked during larval development to induce one of two mutually exclusive recombination events in their germline between either FRT or FRT3. ( B ) Heat shock of hsFLP; hh T2A-QF2-T2A-LexA-GAD and hsFLP; dpp T2A-QF2-T2A-LexA-GAD flies produces frequent recombinants, both T2A-QF2 and T2A-LexA-GAD. The bar graph shows the proportion of heat-shocked animals that produced at least one recombinant offspring. The dot plot shows the proportion of recombinant offspring per heat-shocked parent. Mean ± SD is indicated. ( C–D ) Validation of individual hh T2A-QF2 and hh T2A-LexA-GAD derivatives by immunofluorescence. All panels show third instar larval wing discs dissected from potential hh T2A-QF2-T2A-LexA-GAD recombinants crossed to a QUAS-GFP+LexAop-mCherry reporter line. ( C ) Wing disc from non-recombinant hh T2A-QF2-T2A-LexA-GAD showing expression of both GFP and mCherry in the posterior of the wing disc. Note, this is the same image as shown in . ( D ) Wing disc from recombinant hh T2A-QF2 showing expression of GFP but not mCherry in the posterior of the wing disc. ( E ) Wing disc from recombinant hh T2A-LexA-GAD showing expression of mCherry but not GFP in the posterior of the wing disc. Validation of ( F ) hh T2A-QF2 and hh T2A-LexA-GAD derivatives and ( G ) dpp T2A-QF2 and dpp T2A-LexA-GAD derivatives by PCR from genomic DNA from individual flies. In all panels, for brevity, T2A-QF2-T2A-LexAop, T2A-QF2, and T2A-LexAop, are notated as Q+L, Q(-L), and L(-Q), respectively. Figure 5—source data 1. Original file for the DNA gel analysis in . Figure 5—source data 2. Original file for the DNA gel analysis with boxes indicating the portions cropped to produce the image for .

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Knock-In, Produced, Recombinant, Immunofluorescence, Expressing

Journal: eLife

Article Title: Expanding the Drosophila toolkit for dual control of gene expression

doi: 10.7554/eLife.94073

Figure Lengend Snippet: ( A ) shRNAs for knockdown or genes for overexpression were cloned into pLexAop-WALIUM20 and pQUAS-WALIUM20, derived from the TRiP WALIUM20 vector. ( B–J ) Dorsal view of adult fly thoraces resulting from crosses of LexAop or QUAS shRNAs to da T2A-LexA-GAD (generated in this study), Tub-LexA-GAD (Bloomington Drosophila Stock Center [BDSC] 66686), or Tub-QF2 (BDSC 51958). ( B–C ) white shRNA control produced no thoracic phenotypes in any of the crosses. ( E–G ) forked shRNA produced a forked bristles phenotype (white arrowheads). Note that some bristles retain a more elongated wild-type morphology with the Tub-QF2-driven forked knockdown (G, yellow asterisk). ( H–J ) ebony shRNA produced a darkened cuticle phenotype. The da T2A-LexA-GAD driver produced the strongest phenotype (compare panel H to I and J).

Article Snippet: The digested pUC57 Kan_gw_OK2 backbone, containing the HAs and guides, was then ligated with the digested T2A-LexA/QF2-Hsp70-3xP3-RFP with 2.5 µl 10× T4 DNA ligase buffer (NEB B0202S) and 0.5 µl T4 DNA ligase (NEB M0202S).

Techniques: Knockdown, Over Expression, Clone Assay, Derivative Assay, Plasmid Preparation, Generated, shRNA, Control, Produced