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Matα4 gal vp16

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Matα4-GAL-VP16 is a genetic construct that contains the promoter from the maternal alpha4 tubulin gene fused to the yeast GAL4 transcriptional activator domain and the VP16 activation domain. This construct is used to drive high-level, ubiquitous gene expression in Drosophila.

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Lab products found in correlation

Attp154 strain, by BestGene (2 mentions) Oteb279, by Bloomington Drosophila Stock Center (2 mentions) Otedb, by Bloomington Drosophila Stock Center (2 mentions) Attp2, by Bloomington Drosophila Stock Center (1 mentions) Eyeless gal4, by Bloomington Drosophila Stock Center (1 mentions)

4 protocols using matα4 gal vp16

1

Drosophila Husbandry and Genetic Rescue

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Fly husbandry was conducted according to standard procedures. All crosses were performed at 25°C. The WT strain used was Oregon R. Transgenic lines for expression of UASp-GFP-Ote (WT and mutants) were generated by site-directed insertions of our pUAS-K10attB-based vectors on the third chromosome in the attP154 strain (BestGene). The expression of the UASp-GFP-Ote and UASp-RFP-BAF transgenes in embryos for GFP affinity purification and for video microscopy was driven by matα4-GAL-VP16 (#7062; Bloomington Drosophila Stock Center). For genetic rescue experiments, the expression of UASp-GFP-Ote transgenes was driven by GAL4::VP16-nos.UTR (MVD1) (no. 4937; Bloomington Drosophila Stock Center). Otefin mutant alleles used were oteB279 (no. 16189; Bloomington Drosophila Stock Center) and oteDB (no. 5092; Bloomington Drosophila Stock Center). Fertility tests were done by placing single females with 2–3 Oregon R males in tubes containing grape juice agar and yeast paste at 25°C. Flies were transferred to new tubes every day and the number of eggs laid and percentages of hatched embryos were counted 24 h after removal of the flies from the tubes.
Emond-Fraser V., Larouche M., Kubiniok P., Bonneil É., Li J., Bourouh M., Frizzi L., Thibault P, & Archambault V. (2023). Identification of PP2A-B55 targets uncovers regulation of emerin during nuclear envelope reassembly in Drosophila. Open Biology, 13(7), 230104.
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2

Drosophila RNAi Experiments for Top2 and mei-W68

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Flies were maintained on standard food at 25°C. Fly stocks used for RNAi experiments were y w; spapol, w; {matα4-GAL-VP16}V37 (Bloomington 7063), y1 sc1 v1; P{y[+t7.7] v[+t1.8] = TRiP. GL00338}attP2 (Bloomington 35416) an RNAi construct targeting the Top2 (CG10223) gene, and y v; CG33296 RNAi (described below). To obtain control flies containing only one copy of the driver or RNAi construct, the designated stocks were crossed to y w; spapol flies. Transheterozygotes mutant for mei-W68 (CG7753) were made from the following stocks: y/BS Y; mei-W68Z1049 cn bw/SM6a and y/y+ Y; mei-W68Z4572 cn bw/Cyo[49] (link). The genotype of the nanos-Gal4:VP16 driver flies was y w/y+ Y; nanos-Gal4:VP16; spapol.
Hughes S.E, & Hawley R.S. (2014). Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis. PLoS Genetics, 10(10), e1004650.
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3

Drosophila Husbandry and Genetic Rescue

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Fly husbandry was conducted according to standard procedures. All crosses were performed at 25°C. The WT strain used was Oregon R. Transgenic lines for expression of UASp-GFP-Ote (WT and mutants) were generated by site-directed insertions of our pUAS-K10attB-based vectors on the third chromosome in the attP154 strain (BestGene). The expression of the UASp-GFP-Ote and UASp-RFP-BAF transgenes in embryos for GFP affinity purification and for video microscopy was driven by matα4-GAL-VP16 (#7062; Bloomington Drosophila Stock Center). For genetic rescue experiments, the expression of UASp-GFP-Ote transgenes was driven by GAL4::VP16-nos.UTR (MVD1) (no. 4937; Bloomington Drosophila Stock Center). Otefin mutant alleles used were oteB279 (no. 16189; Bloomington Drosophila Stock Center) and oteDB (no. 5092; Bloomington Drosophila Stock Center). Fertility tests were done by placing single females with 2–3 Oregon R males in tubes containing grape juice agar and yeast paste at 25°C. Flies were transferred to new tubes every day and the number of eggs laid and percentages of hatched embryos were counted 24 h after removal of the flies from the tubes.
Emond-Fraser V., Larouche M., Kubiniok P., Bonneil É., Li J., Bourouh M., Frizzi L., Thibault P, & Archambault V. (2023). Identification of PP2A-B55 targets uncovers regulation of emerin during nuclear envelope reassembly in Drosophila. Open Biology, 13(7), 230104.
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4

Polo Kinase Regulation in Drosophila

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Fly husbandry was conducted according to standard procedures. All crosses were performed at 25 or 27 °C. The WT strain used was Oregon R. Transgenic flies for expression of UAS-Polo-GFP (WT and mutants) were created by site-directed insertions after injections of our plasmids (pUAS-K10attB-based) in the attP154 strain by BestGene. Insertions on the third chromosome allowing very similar expression levels were selected for experiments (PhiC31 integrase-mediated site-specific transgenesis, BestGene Inc). The UAS-Polo RNAi strain used for depletion of Polo was obtained from Vienna Drosophila Resource Center (#20177). Expression of Polo transgenes in the early embryo was driven by matα4-GAL-VP16 (#7062, Bloomington Drosophila Stock Center). Expression of Polo transgenes and depletion of endogenous Polo in the developing head tissues was driven by eyeless-Gal4 (#5534, Bloomington Drosophila Stock Center).
For fertility tests, well-fed females were mixed with males in tubes containing grape juice agar and allowed to lay eggs for 1 day before being removed. The percentage of hatched embryos was counted 24 h later.
For viability tests, flies were crossed and the number of observed flies (number of pupae hatching) relative to their expected number in the progeny (total number of pupae) was expressed as a percentage.
Kachaner D., Garrido D., Mehsen H., Normandin K., Lavoie H, & Archambault V. (2017). Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry. Nature Communications, 8, 1701.
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