Fly Facility

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Fly Facility


The fruit fly, Drosophila melanogaster has been an influential invertebrate model organism in understanding gene function for over a century. In addition to being suitable for classical genetics, its adaptability to evolving gene modification tools ranging from transgenesis to novel genome modification tools including CRISPR has made the fly a sought after model organism in modern biology. Over the past five years the Fly Facility, C-CAMP has provided services to facilitate modern molecular genetics in Drosophila.

Drosophila Transgenesis

 

CRISPR-Cas9 based genomic editing

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Drosophila Transgenesis:

Give us a vial of DNA and we will give you a vial of flies with the DNA integrated! Drosophila transgenesis is a labour intensive process and requires optimised protocols and technical expertise. This process often diverts attention from the core research project. We are offering Drosophila Transgenesis services at competitive prices. We are offering both P-element mediated and PhiC31-integrase mediated transgenesis services. With the state-of-art equipment and the experienced staff we are able to generate transgenics in throughput rate for large scale projects.

a) P-element Mediated Transgenesis:

  • P-element Classic - Injection of DNA into the embryos, raising the survived larvae to adulthood and crossing the G0 flies (>100 individual crosses). Identification and collection of transformants; the collected transformant flies (at least 3 independent lines) are sent to the customer and original vials are maintained in the facility for two weeks as backup.

 

  • P-element Excel - This involves mapping the insertions of the screened transgenic lines followed by the Classic service and balancing the insertions.

b) PhiC31-integrase Mediated Transgenesis:

  • Site-Specific Excel - Injection of DNA into the embryos of PhiC31 integrase & selected attP docking site (click here and choose attP sites), crossing of G0 flies, screening the transformants and balancing the insertions. 

 

  • Site-Specific BAC- This service includes injecting the BACs or fosmids (>30kb) into 500 embryos of strain (or a cross of) PhiC31-integrase and a docking site of the user’s choice and scoring the transformants, balancing and shipping. 

 

  • Site-Specific CRISPR- This service includes injecting the plasmids with CRISPR/Cas9 guide RNA into 300 embryos of strain (or a cross of) PhiC31-integrase and a docking site of the user’s choice and scoring the transformants, balancing and shipping.                                                                                                 

CRISPR-Cas9 based genomic editing:

 

 

Recently, the fly facility has started using CRISPR-Cas9 system to generate deletions.  The use of the CRISPR-Cas9 nuclease system is a very young but rapidly developing area of modern biology. The type II CRISPR system from S. pyogenes has been adapted for inducing sequence-specific DSBs and targeted genome editing. In this system, Cas9 endonuclease and a target sequence specific guide RNA (gRNA) are introduced into a heterologous system to generate DSBs in their genome in a sequence specific manner, making it a useful genetic engineering tool to induce deletions, insertions, and precisely defined modifications in genomes of diverse organisms.

This technique has been used in Drosophila to mutate about a dozen genes and different methods to improve efficiency are being actively under research. Our facility is providing the service of sgRNA injections in various fly backgrounds in addition to full genomic deletion service. Please write to us for further details.

 

 

Large Scale Projects:

Establishment of a genome wide set of tagged fosmid reagents for the analysis of gene function in Drosophila melanogaster:

Fly Facility in collaboration with Pavel Tomancak, Frank Scnorrer, Mihail Sarov and other collaborators at MPI-CBG, Dresden and K. VijayRaghavan’s & Mani Ramaswami’s  groups at NCBS is involved in generating transgenics for a large scale project “TransgeneOme”. So far, Fly Facility has generated about 500 transgenic lines for the genomic clones of size ranging from 35-55kb by PhiC31 integrase mediated transgenesis.

The project aims at expansion of the spectrum of available genomic resources by creating a comprehensive set of tagged proteins within their native genomic regulatory context. These reagents will enable both the visualization of the tissue specificity and sub-cellular localization of proteins throughout the Drosophila life cycle and purification of protein-protein or protein-DNA complexes in a tissue specific manner.

Drosophila protein interaction map (DPiM):

Fly Facility has also generated about 5000 transgenic lines, for another large scale project DPiM, by injecting fly embryos with plasmid construct containing the ORFs fused to a HA tag sequence.

The goal of this project is to study how proteins encoded by Drosophila cDNAs interact with each other to form functional complexes. Out of the 13600 genes in the fly genome identified, ~5000 genes are fused to a HA tag to generate stable transgenic flies, from which the HA fused bait protein would be recovered along with its interacting partners, by Tandem Affinity Purification. The resulting protein complex would be studied through mass spectroscopic analysis, to identify the interacting partner. The expression of the bait protein in these flies is driven by a UAS promoter to allow spatio-temporal regulation.

 

 

For more information on Fly (Drosophila) Facility, please write to services [at] ccamp.res.in or flyfacility [at] ccamp.res.in

Deepti Trivedi, In-Charge Fly Facility.