Cell type-specific manipulation with GFP-dependent Cre recombinase.

Nature neuroscience (2015-08-11)
Jonathan C Y Tang, Stephanie Rudolph, Onkar S Dhande, Victoria E Abraira, Seungwon Choi, Sylvain W Lapan, Iain R Drew, Eugene Drokhlyansky, Andrew D Huberman, Wade G Regehr, Constance L Cepko
ABSTRACT

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Magnesium chloride, anhydrous, ≥98%
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Sodium pyruvate, ReagentPlus®, ≥99%
Sigma-Aldrich
(+)-Sodium L-ascorbate, crystalline, ≥98%
Sigma-Aldrich
Choline chloride, ≥98%
Sigma-Aldrich
Choline chloride, ≥99%
Sigma-Aldrich
Sodium pyruvate, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99%
Sigma-Aldrich
Sodium bicarbonate, BioXtra, 99.5-100.5%
Sigma-Aldrich
(+)-Sodium L-ascorbate, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
(+)-Sodium L-ascorbate, BioXtra, ≥99.0% (NT)
Sigma-Aldrich
Magnesium chloride, powder, <200 μm
Sigma-Aldrich
Choline chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥98%
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Magnesium chloride solution, BioUltra, for molecular biology, 2 M in H2O
Sigma-Aldrich
Magnesium chloride, BioReagent, suitable for insect cell culture, ≥97.0%
Sigma-Aldrich
Sodium pyruvate, powder, BioXtra, suitable for mouse embryo cell culture
Sigma-Aldrich
Potassium D-gluconate, ≥99%
Sigma-Aldrich
Magnesium chloride solution, PCR Reagent, 25 mM MgCI2 solution for PCR
Sigma-Aldrich
Potassium hydroxide, anhydrous, ≥99.95% trace metals basis
Sigma-Aldrich
Sodium pyruvate, BioXtra, ≥99%
Sigma-Aldrich
Potassium gluconate, meets USP testing specifications, anhydrous
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium bicarbonate, Hybri-Max, powder, suitable for hybridoma, ≥99.5%
Sigma-Aldrich
Sodium pyruvate, Hybri-Max, powder, suitable for hybridoma
Sigma-Aldrich
Choline chloride, BioUltra, ≥99.0% (AT)
Sigma-Aldrich
Magnesium chloride, AnhydroBeads, −10 mesh, 99.9% trace metals basis
Sigma-Aldrich
Sodium pyruvate, anhydrous, free-flowing, Redi-Dri, ReagentPlus®, ≥99%