An efficient method for recombineering GAL4 and QF drivers

Abstract

IntroductionA current challenge in neuroscience is to develop connectomes, maps of the functional connections between neurons in a nervous system. A set of GAL4 and QF drivers specific for neurotransmitters and neurotransmitter receptors would facilitate the development of a Drosophila connectome since knowledge of the neurotransmitter usage of a presynaptic neuron entails knowledge of the neurotransmitter receptor usage of its post-synaptic neuron and vice versa. In Drosophila, many neurotransmitter-specific genes, such as neurotransmitter synthesis enzymes, and most neurotransmitter receptors have extensive conserved noncoding regions (presumably regulatory) that are too large to be contained in traditional plasmid vectors suitable for generating GAL4 and QF drivers. [1][2][3] Therefore, generation of GAL4 and QF drivers that faithfully recapitulate the entire endogenous spatial and temporal expression patterns of neurotransmitter and neurotransmitter receptor-specific genes will require a different type of vector capable of maintaining larger DNA fragments. The recently developed Drosophila genomic CH321 bacterial artificial chromosome (BAC) library has an average insert size of 83 kb 4 and therefore has much greater potential for including in a single clone the complete regulatory region of large Drosophila genes including neurotransmitter synthesis enzymes and neurotransmitter receptors. Since BACs of this size are too large for traditional restriction enzyme cloning, modifying them into GAL4 and QF drivers requires recombineering. In this report an efficient, generally applicable method for recombineering GAL4 and QF cassettes into BACs using kanamycin selection is functionally demonstrated.Neural circuit mapping and manipulation are facilitated by independent control of gene expression in pre-and postsynaptic neurons. the GaL4/Uas and Q binary transcription systems have the potential to provide this capability. Of particular use in neural circuit mapping would be GaL4 and QF drivers specific for neurotransmitters and neurotransmitter receptors. Recently available Drosophila genomic Bac libraries make recombineering large genes including those specific for neurotransmitters and neurotransmitter receptors feasible. here the functionality of cassettes that allow efficient recombineering of GaL4 and QF drivers based on kanamycin selection is demonstrated in Drosophila. the cassettes should, however, be generalizable for recombineering in other species.An efficient method for recombineering GAL4 and QF drivers Keywords: GAL4, QF, recombineering, tyramine beta-hydroxylase, tyramine receptor, bacterial artificial chromosome ResultsOne method for recombineering genes of interest into BACs utilizes a two-step process that involves positive selection for galK in the first step followed by replacement of galK with the gene of interest using negative selection in the second step. 5 Twentytwo BACs from the recently generated Drosophila genomic BAC libraries 4 containing either neurotransmitter synthesis enzymes or neu...

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

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Serial Recombineering Cloning to Build Selectable and Tagged Genomic P[acman] BAC Clones for Selection Transgenesis and Functional Gene Analysis using Drosophila melanogaster

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Developmental loss of neurofibromin across distributed neuronal circuits drives excessive grooming in Drosophila

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