http://genetics.wustl.edu/bio5491/files/2012/02/Pan_Yorkie_Cell.pdf

The Hippo Signaling Pathway Coordinately Regulates Cell Proliferation and Apoptosis by Inactivating Yorkie, the Drosophila Homolog of YAP

Or83b encodes a broadly expressed odorant receptor essential for Drosophila olfaction.

We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.

https://www.genetics.org/content/genetics/194/4/1029.full.pdf

Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease

Gene targeting—the process of gene replacement by homologous recombination—is a very useful but typically inefficient technique ( [1][1] ). A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a

http://science.sciencemag.org/content/sci/300/5620/764.full.pdf

Enhancing gene targeting with designed zinc finger nucleases.

https://www.cell.com/cell-reports/pdf/S2211-1247(13)00312-4.pdf

Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System

Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting Both have been used for targeted mutagenesis, but require donors of differing design Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus Although ends-in targeting has been successful in Drosophila, an attempt at ends-out targeting failed To test whether ends-out targeting could be used in Drosophila, we applied two strategies for ends-out gene replacement at the endogenous yellow (y) locus in Drosophila First, a mutant allele was rescued by replacement with an 8-kb y+ DNA fragment at a rate of ≈1/800 gametes Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of ≈1/380 gametes The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting

https://www.pnas.org/content/pnas/100/5/2556.full.pdf

Ends-out, or replacement, gene targeting in Drosophila

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.

https://www.genetics.org/content/genetics/172/1/275.full.pdf

Loss of Hsp70 in Drosophila Is Pleiotropic, With Effects on Thermotolerance, Recovery From Heat Shock and Neurodegeneration

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned ∼47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.

https://www.genetics.org/content/genetics/168/3/1467.full.pdf

Genomic Deletions of the Drosophila melanogaster Hsp70 Genes

We present detailed protocols for two methods of gene targeting in Drosophila. The first, ends-out targeting, is identical in concept to gene replacement techniques used routinely in mammalian and yeast cells. In Drosophila, the targeted gene is replaced by the marker gene white + (although options exist to generate unmarked targeted alleles). This approach is simple in both the molecular cloning and the genetic manipulations. Ends-out will likely serve most investigators' purposes to generate simple gene deletions or reporter gene "knock-ins." The second method, ends-in targeting, targets a wild-type gene with an engineered mutated copy and generates a duplication structure at the target locus. This duplication can subsequently be reduced to one copy, removing the wild-type gene and leaving only the introduced mutation. Although more complicated in the cloning and genetic manipulations (see Note 1), this approach has the benefit that the mutations may be introduced with no other remnant of the targeting procedure. This "surgical" approach will appeal to investigators who desire minimal perturbation to the genome, such as single nucleotide mutation. Although both approaches appear to be approximately equally efficient (see Note 2), each method has separate strengths and drawbacks. The choice of which approach is best depends on the researcher's goal.

Methods for homologous recombination in Drosophila.

A gene targeting method for use in a host organism whereby the host organism is transfected with an ends-out donor construct. Further transfecting the host organism with two transgenes expressing endonuclease and recombinase enzymes. The endonuclease and recombinase enzymes are used so that homologous recombination occurs between the DNA segment of the donor construct and a selected gene of the host organism, thus forming a host with containing the recombinogenic donor. The progeny of the host organism, which include the recombinogenic donor are then selected.

Wei J. Gong

Papers

Ends-out, or replacement, gene targeting in Drosophila

Loss of Hsp70 in Drosophila Is Pleiotropic, With Effects on Thermotolerance, Recovery From Heat Shock and Neurodegeneration

Genomic Deletions of the Drosophila melanogaster Hsp70 Genes

Methods for homologous recombination in Drosophila.

Ends-out gene targeting method