Gene Activation Using FLP Recombinase in C. elegans

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an “off cassette” between the promoter and the coding region of the desired product. The “off cassette” is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner.

[1]  H. Horvitz,et al.  The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins. , 1994, Genetics.

[2]  S. Dymecki.  Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Hartley,et al.  Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones. , 2004, Genome research.

[4]  J. Thomas,et al.  Genetic analysis of defecation in Caenorhabditis elegans. , 1990, Genetics.

[5]  M. Chalfie,et al.  Targeted cell killing by reconstituted caspases , 2007, Proceedings of the National Academy of Sciences.

[6]  Erik M. Jorgensen,et al.  The Sensory Circuitry for Sexual Attraction in C. elegans Males , 2007, Current Biology.

[7]  H. Horvitz,et al.  The GABAergic nervous system of Caenorhabditis elegans , 1993, Nature.

[8]  J. Hartley,et al.  DNA cloning using in vitro site-specific recombination. , 2000, Genome research.

[9]  E. Bamberg,et al.  Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses , 2005, Current Biology.

[10]  D. Butterfield,et al.  Oxidative stress precedes fibrillar deposition of Alzheimer’s disease amyloid β-peptide (1–42) in a transgenic Caenorhabditis elegans model , 2003, Neurobiology of Aging.

[11]  R. Holmgren,et al.  Expression of the Drosophila gooseberry locus defines a subset of neuroblast lineages in the central nervous system. , 1995, Developmental biology.

[12]  K. Golic,et al.  Local transposition of P elements in Drosophila melanogaster and recombination between duplicated elements using a site-specific recombinase. , 1994, Genetics.

[13]  N. Henderson,et al.  Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome. , 1989, Nucleic acids research.

[14]  Feng Zhang,et al.  Multimodal fast optical interrogation of neural circuitry , 2007, Nature.

[15]  F. Benfenati,et al.  Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin , 1992, Nature.

[16]  N. Henderson,et al.  Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[17]  T. Südhof,et al.  SNARE Function Analyzed in Synaptobrevin/VAMP Knockout Mice , 2001, Science.

[18]  D. Dixon,et al.  Differential regulation of closely related members of the hsp16 gene family in Caenorhabditis elegans. , 1989, DNA.

[19]  S. Lindquist,et al.  The FLP recombinase of yeast catalyzes site-specific recombination in the drosophila genome , 1989, Cell.

[20]  M. Chalfie,et al.  Combinatorial Marking of Cells and Organelles with Reconstituted Fluorescent Proteins , 2004, Cell.

[21]  David E Hill,et al.  A first version of the Caenorhabditis elegans Promoterome. , 2004, Genome research.

[22]  S. Shaham,et al.  Temporal Control of Cell-Specific Transgene Expression in Caenorhabditis elegans , 2007, Genetics.

[23]  E. Jorgensen,et al.  EXP-1 is an excitatory GABA-gated cation channel , 2003, Nature Neuroscience.

[24]  S. Mango,et al.  Regulation of Organogenesis by the Caenorhabditis elegans FoxA Protein PHA-4 , 2002, Science.

[25]  J. Hudson,et al.  C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression , 2003, Nature Genetics.

[26]  H. Horvitz,et al.  Genes required for GABA function in Caenorhabditis elegans , 1993, Nature.