Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

Abstract

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a "Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated" (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

Introduction

The genetic toolbox for targeted gene expression has been well developed in Drosophila, making it one of the best model systems to investigate the function of genes involved in a wide variety of cellular processes. Binary expression systems, such as yeast Gal4/UAS (upstream activation sequence), was first adopted for tissue-specific enhancer trapping and gene misexpression in the Drosophila genetic model1 (Figure 1). This system facilitated the development of a large number of techniques such as spatiotemporal regulation of gene overexpression, misexpression, knockout in selected groups o....

Protocol

1. Designing and Constructing the gRNA Expression Vector

  1. To precisely replace a long defined region of an exon, use a dual gRNA approach6, in which each gRNA can specifically target two ends of the selected region of interest. To obtain an accurate gene-specific spatiotemporal expression of the driver, select two gRNA target sites within the first coding exon of the gene.
  2. For Drosophila melanogaster, select the gRNA target sites using the flyCRISPR Optimal Ta.......

Representative Results

This protocol was successfully used to generate a targeted binary expression reporter system specific for bnl expressing cells5. The cis-regulatory elements (CREs) that control complex spatiotemporal bnl expression are not characterized. Therefore, to achieve spatiotemporal expression under the control of the endogenous bnl regulatory sequence, only the first coding exon of bnl was designed to be replaced with the sequen.......

Discussion

Traditionally, Drosophila enhancer traps were generated by two different methods. One of the ways includes random insertion of a driver (eg., Gal4) sequence in the genome by transposition (e.g., P-element transposition)1 . Alternatively, the driver sequences can be placed under the transcriptional control of a putative enhancer/promoter region in a plasmid construct, which would then be integrated into an ectopic site of the genome3,

Acknowledgements

We thank Dr. F. Port, Dr. K. O'Connor-Giles, and Dr. S. Feng for discussions on CRISPR strategy; Dr. T.B. Kornberg, and the Bloomington Stock Center for reagents; UMD imaging core facility; and funding from NIH: R00HL114867 and R35GM124878 to SR.

....

Materials

NameCompanyCatalog NumberComments
X-Gal/IPTGGentrox (Genesee Scientific)18-218cloning
LB-AgarBD DifcoBD 244520cloning
Tris-HClSigma AldrichT3253Molecular Biology
EDTASigma AldrichE1161Molecular Biology
NaClSigma AldrichS7653Molecular Biology
UltraPure DNase/RNase-Free WaterThermoFisher Scientific10977-023Molecular Biology
10%SDSSigma Aldrich71736Molecular Biology
KOAcFisher-ScientificP1190Molecular Biology
EtOHFisher-Scientific04-355-451Molecular Biology
GeneJET MiniprepThermoFisher ScientificK0503Miniprep
PureLink HiPure Plasmid Maxipep kitsThermoFisher ScientificK210006Maxiprep
BbsINEBR0539SRestriction enzyme
PrimersIDT-DNAPCR
pCFD4Kornberg LabDNA template and vector for gRNA
KAPA HiFi Hot Start- (Kapa Biosystems)Kapa biosystemsKK2601PCR
Q5-high fidelity TaqNEBNEB #M0491PCR
Gibson Assembly Master MixNEBNEB #E2611DNA assembly
pBPnlsLexA:p65UwAddgeneDNA template for LexA amplification
Proteinase KThermoFisher Scientific25530049Molecular Biology
2x PCR PreMix, with dye (red)SydlabMB067-EQ2RMolecular Biology
Gel elution kitZymo Research (Genesee Scientific)11-300Molecular Biology
TRI reagentSigma-AldrichMolecular Biology
Direct-zol RNA purification kitsZymo Research (Genesee Scientific)11-330Molecular Biology
OneTaq One-Step RT-PCR KitNEBE5315SMolecular Biology
lexO-CherryCAAXKornberg LabFly line
UAS-CD8:GFPKornberg labFly line
btl-Gal4Kornberg labFly line
MKRS/TB6BKornberg labFly line
Confocal Microscope SP5XLeicaImaging expression pattern
CO2 stationGenesee Scientific59-122WCUfly pushing
Stereo microscopeOlympusSZ-61fly pushing
Microtube homogenizing pestlesFisher-Scientific03-421-217genomic DNA isolation
NanoDrop spectrophotometerThermoFisher ScientificND-1000DNA quantification

References

  1. Brand, A. H., Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118 (2), 401-415 (1993).
  2. Lai, S. -. L., Lee, T. Geneti....

Explore More Articles

Tissue specificBinary Transcription SystemsDrosophilaGenome EditingCRISPR Cas9FGF HomologBranchlessTracheal Airway EpitheliumSpatiotemporal ExpressionTrans ActivatorGAL4LexAUASLexOEndogenous RegulationGuide RNAPCFD4 Vector

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved