Recombineering Homologous Recombination Constructs in Drosophila

Summary

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila⁶. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Abstract

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Introduction

Clean molecularly-defined manipulations of single genes at their endogenous loci offer an invaluable tool to study a myriad of questions relevant to eukaryotic biology. Drosophila reverse genetic techniques for generating loss-of-function alleles had proven to be challenging until Golic and colleagues introduced in vivo gene targeting using homologous recombination to Drosophila ^1-3. They demonstrated that specific genomic loci could be targeted using a linear fragment of DNA from an integrated transgenic construct. This linear "donor" DNA is generated in vivo through FRT-mediated recombination (to excise the DNA from the....

Protocol

1. Selection of BAC and Region to Target

1. To acquire a BAC with the gene of interest (GOI) (here CG32095 is used as an example), search for CG32095 at www.flybase.org. Under the section stocks and reagents, check the section entitled, "Genomic Clones" for BACs containing CG32095. Make sure that the BAC includes at least 10 kb upstream and 5 kb downstream the gene of interest. Alternatively, clones in the P[acman] vector⁷ can also be found at http://www.pacmanfly.org/libraries.html.
2. The BAC....

Results

Amplification of the LA and RA homology arms should produce 500 bp products and the PCR-SOE reaction should yield a 1.0 kb product (Sections 2.1-2.4; Figure 2). The BP reaction performed in section 2.5 is typically very efficient and bacterial transformation of the product yields 5-100 colonies on average. Nearly all the colonies tested with PCR check show the expected PCR product.

During the first round of recombineering (Section 3) expect to get 20-40 colonies after transfor.......

Discussion

The power of genetic model organisms in biomedical research is largely based on the tools available for genetic manipulation. The small models C. elegans and Drosophila in particular allow for inexpensive and fast molecular genetic analyses of complete pathways and gene families implicated in multicellular development or function. Recent years have seen significant advances in tool development for manipulating genes in Drosophila ^14,15. For example, recombineering, which is widely us.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SW102 Recombination competent bacteria	NCI-Frederick	Recombination Bacteria (SW102, SW105 and SW106)	http://ncifrederick.cancer.gov/research/brb/logon.aspx
TransforMax EPI300 electrocopmpetent E. coli	Epicentre	EC300110	Includes CopyControl induction solution
PfuUltra II Fusion HS DNA Polymerase	Aligent Technology Inc.	600670
BamHI-HF	New England Biolabs	R3136S
Zymoclean Gel DNA Recovery Kit	Zymo Research	D4001
Use Gateway BP ClonaseII Enzyme kit	Invitrogen	11789-020
P[acman]KO1.010	Buszczak and Hiesinger Labs	Upon request
pENTR RFP-Kan11	Buszczak and Hiesinger Labs	Upon request
Flystocks	Bloomington stock center	Stock numbers: 25680, 25679	y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B snaSco/CyO, P{hs-hid}4 y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}23 P{70I-SceI}4A/TM3, P{hs-hid}14, Sb1
Electroporation machine	Biorad GenePulser Xcell with PC module	165-2662
Cuvettes	Fisher Brand	#FB101