Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80

doi: 10.1073/pnas.2304730120

Figure Lengend Snippet: Schematic comparison of Gal4, split-Gal4, and split-intein Gal4. ( A ) The Gal4 transcription factor binds to UAS sequences to drive transcription and can be repressed by the binding of Gal80. Gal4 is drawn here as a monomer, but functions as a dimer in vivo. ( B ) In the original split-Gal4 system, the Gal4DBD and a strong transcriptional activator (VP16 or p65) are each driven by separate enhancers and reconstituted in cells by leucine zipper domains. Gal80 cannot bind or repress the split-Gal4 complex. ( C ) In the split-intein Gal4 system, two fragments of the Gal4 protein, each flanked by a split-intein, are independently driven by separate enhancers and seamlessly transspliced to reconstitute a functional, wild-type Gal4 protein, which can be repressed by Gal80.

Article Snippet: To test the transcriptional activation strength of each system in cell culture, we transiently transfected either split-intein components (Gal4 N-int and Gal4 C-int ) or NanoTag Split-Gal4 components (Gal4DBD-3xNb12701 and Gal4AD-1x127D01), all driven by a constitutive Actin5c promoter, into S2R+ cells, along with a green fluorescent protein reporter (UAS:GFP).

Techniques: Binding Assay, In Vivo, Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80

doi: 10.1073/pnas.2304730120

Figure Lengend Snippet: Mapping scRNA clusters to anatomy using split-intein Gal4 lines based on NS-Forest v2 predictions. Based on a gut-specific scRNA dataset from Hung et al. , the NS-Forest v2 algorithm identified pairs of genes to mark specific clusters in the adult gut. Left: Summed expression in each of the 22 clusters is shown on the Left . Right : In vivo expression of split-intein Gal4 lines in adult guts. Brackets indicate the approximate position of the copper and iron cell (Cu–Fe) region. ( A ) Peritrophin-15a ∩ CG4830 drives expression in a band of enterocytes anterior to the copper cells. ( B ) CG43774 ∩ thetaTry drives expression in the copper and iron cells. ( C ) LManV ∩ ninaD drives expression in a posterior band of enterocytes. Anterior is to the left.

Article Snippet: To test the transcriptional activation strength of each system in cell culture, we transiently transfected either split-intein components (Gal4 N-int and Gal4 C-int ) or NanoTag Split-Gal4 components (Gal4DBD-3xNb12701 and Gal4AD-1x127D01), all driven by a constitutive Actin5c promoter, into S2R+ cells, along with a green fluorescent protein reporter (UAS:GFP).

Techniques: Expressing, In Vivo

Journal: bioRxiv

Article Title: split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80

doi: 10.1101/2023.03.24.534001

Figure Lengend Snippet: (A) Cartoon schematic of the split-intein GeneSwitch system, not drawn to scale. The same N-terminal fragment of Gal4 used in split-intein Gal4 can be combined with the C-terminus of the GeneSwitch system, which includes amino acids 21–93 of Gal4, a progesterone ligand binding domain (PR-LBD) and the p65 transcriptional activator. (B) Drug-inducible ISC tumor model using the esg -Gal4 N-int ∩ tub -GeneSwitch C-int > UAS: yki S3A . Anterior is up. Scale bar = 50 µm.

Article Snippet: The inserts for the original split-Gal4 components (pAW-Zip − -Gal4DBD and pAW-p65-Zip + ) were amplified from pBPZpGAL4DBDUw (Addgene 26233) and pBPp65ADZpUw (Addgene 26234), respectively. pCaSpeR-tub-Gal80 was used to test for Gal80 sensitivity in S2R+ cells.

Techniques: Ligand Binding Assay

Journal: bioRxiv

Article Title: split-intein Gal4 provides intersectional genetic labeling that is fully repressible by Gal80

doi: 10.1101/2023.03.24.534001

Figure Lengend Snippet: (A) The Gal4 transcription factor binds to UAS sequences to drive transcription, and can be repressed by the binding of Gal80. Gal4 is drawn here as a monomer, but functions as a dimer in vivo. (B) In the original split-Gal4 system, the Gal4DBD and a strong transcriptional activator (VP16 or p65) are each driven by separate enhancers, and reconstituted in cells by leucine zipper domains. Gal80 cannot bind or repress the split-Gal4 complex. (C) In the split-intein Gal4 system, two fragments of the Gal4 protein, each flanked by a split-intein, are independently driven by separate enhancers, and seamlessly trans-spliced to reconstitute a functional, wildtype Gal4 protein, which can be repressed by Gal80.

Article Snippet: The inserts for the original split-Gal4 components (pAW-Zip − -Gal4DBD and pAW-p65-Zip + ) were amplified from pBPZpGAL4DBDUw (Addgene 26233) and pBPp65ADZpUw (Addgene 26234), respectively. pCaSpeR-tub-Gal80 was used to test for Gal80 sensitivity in S2R+ cells.

Techniques: Binding Assay, In Vivo, Functional Assay