Toolbox/Paper 4

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Contents


Paper 4 Model Systems

Drosophila nervous system

Paper 4 Techniques

Homologous recombination in Drosophila

Homologous recombination is a technique that allows you to replace an allele with an engineered construct while not affecting any other loci in the genome. In the Manoli et al paper, homologous recombination was used to generate the fruP1-Gal4 construct.

Procedure: design the DNA construct that you want to insert --> flank this construct with DNA sequences identical to the sequences in the target locus (so that your construct can later find the homologous chromosome during mitosis/meiosis) --> add your engineered construct to cells containing the targeted gene of interest --> during mitosis/meiosis, homologous chromosomes will align and your engineered construct can find the targeted gene --> recombination takes place (switching of the target gene with your engineered construct) --> end result is the altered targeted locus, while the rest of the genome remains unchanged

Inverted repeats

Double-stranded RNA (RNA-mediated interference) has become a standard tool used to silence post-transcriptional expression of a single gene, and the results can provide some insight on the gene’s function. dsRNA can be induced using inverted repeats, which consist of two arms of DNA that surround a spacer region. Inverted repeats have been associated with genomic instability, and are also known to induced homologous recombination.

Male-male habituation

Male fruit flies will court other male flies when exposed to them, initially. Over time, they learn not to court each other. This is called male-male habituation, or "Experience-Dependent Courtship Modification". (Vaias, et al. 1993) The courtship displayed towards other males is the essentially the same courtship behavior displayed towards other females: the behavior is identical for courtship of either sex, intially. The Vaias paper demonstrates that exposure to pheromones released by young males causes male-male habituation. In the discussion paper 'Baker, et al. 2005' on the fruitless gene, they inhibited the expression of Fru-M in olfactory neurons with UAS-Fru-M- IR, and observed that the inhibited males courted males far longer than males with a control transgene.

Courtship conditioning

Recently mated females display a rejection response to males trying to court them. In courtship conditioning, males learn to no longer attempt to court these females, and have a longer courtship delay time in approaching virgin females after exposure to a mated female. In the Fruitless paper (Baker, et al. 2005), they found that the inhibition by UAS-fruM-IR of Fru-M expression in mushroom body neurons decreased any conditioning caused by exposure to mated females. Note: Mated females reject males by a behavior called ovipositor extrusion. However, masculinization of fruP1-GAL4-expression neurons and FruM expression in females led the mutants to show decreased levels of ovipositor extrusion (see fig. 4d of Manoli, et al. 2005).

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