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Simplified Insertion of Transgenes Onto Balancer Chromosomes via Recombinase-Mediated Cassette Exchange.
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PMID:  22670225     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Balancer chromosomes are critical tools for Drosophila genetics. Many useful transgenes are inserted onto balancers using a random and inefficient process. Here we describe balancer chromosomes that can be directly targeted with transgenes of interest via recombinase-mediated cassette exchange (RMCE).
Authors:
Florence F Sun; Justine E Johnson; Martin P Zeidler; Jack R Bateman
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Publication Detail:
Type:  Journal Article     Date:  2012-05-01
Journal Detail:
Title:  G3 (Bethesda, Md.)     Volume:  2     ISSN:  2160-1836     ISO Abbreviation:  G3 (Bethesda)     Publication Date:  2012 May 
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Created Date:  2012-06-06     Completed Date:  2012-09-10     Revised Date:  2013-05-30    
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Nlm Unique ID:  101566598     Medline TA:  G3 (Bethesda)     Country:  United States    
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Languages:  eng     Pagination:  551-3     Citation Subset:  -    
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Journal Information
Journal ID (nlm-ta): G3 (Bethesda)
Journal ID (iso-abbrev): Genetics
Journal ID (hwp): ggg
Journal ID (pmc): ggg
Journal ID (publisher-id): ggg
ISSN: 2160-1836
Publisher: Genetics Society of America
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Copyright © 2012 Sun et al.
open-access:
Received Day: 01 Month: 2 Year: 2012
Accepted Day: 02 Month: 3 Year: 2012
Electronic publication date: Day: 1 Month: 5 Year: 2012
collection publication date: Month: 5 Year: 2012
Volume: 2 Issue: 5
First Page: 551 Last Page: 553
ID: 3362938
PubMed Id: 22670225
Publisher Id: GGG_002097
DOI: 10.1534/g3.112.002097

Simplified Insertion of Transgenes Onto Balancer Chromosomes via Recombinase-Mediated Cassette Exchange
Florence F. Sun*
Justine E. Johnson*
Martin P. Zeidler
Jack R. Bateman*1
*Biology Department, Bowdoin College, Brunswick, Maine 04011
MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Sheffield S10 2TN, United Kingdom
Supporting information is available online at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.112.002097/-/DC1
1Corresponding author: Biology Department, Bowdoin College, 6500 College Station, Brunswick, ME 04011. E-mail: jbateman@bowdoin.edu

In Drosophila, balancer chromosomes bearing multiple inversions are routinely used in genetic manipulations and in the maintenance of sterile or lethal mutations as balanced heterozygotes. Balancer chromosomes typically carry dominant markers, the most common of which affect adult structures only. However, using transgenic approaches, many new markers and functions have been assigned to balancers in efforts to improve their utility. For example, transgenic insertions have been created to facilitate the identification of balanced progeny at different stages of development, including balancers that carry histological or fluorescent markers driven by embryonic enhancers (including so-called “blue” and “green” balancers) (Casso et al. 2000; Halfon et al. 2002; Le et al. 2006; Panzer et al. 1993; Rudolph et al. 1999). Balancers carrying transgenic insertions of GAL80, a repressor of the UAS/GAL4 system, function similarly in cross schemes involving transgenes driven by UAS (Vef et al. 2006). More recently, the cloning of the gene responsible for Tubby1, a convenient marker that is visible during larval development and is carried on the third chromosome balancer TM6B, has led to the creation of Tubby1 transgenes inserted onto X and second chromosome balancers (Guan et al. 2006; Lattao et al. 2011; Pina and Pignoni 2012). In addition to novel markers for the identification of balanced progeny, others have created transgenic insertions on balancer chromosomes for the convenient delivery of key enzymes into genetic schemes; these include transposases for P (Lindsley and Zimm 1992) and Minos (Metaxakis et al. 2005) transposon systems, and Cre (Siegal and Hartl 1996) and FLP (Chou and Perrimon 1992) recombinases. Furthermore, autosomal balancers have been engineered to carry the cell death promoter hid in an effort to simplify fly sorting during gene replacement by homologous recombination (Huang et al. 2008). Thus, a pattern exists in which the development of new genetic technologies consistently leads researchers to target new transgenes to Drosophila balancer chromosomes.

For each of the examples listed above, transgenic insertions were incorporated onto balancers using P-element–mediated transgenesis (Rubin and Spradling 1982). P-element insertion occurs in an untargeted manner; thus, obtaining transgenes on a balancer requires one to create many independent insertions, and then to screen for those that happened to insert onto the balancer chromosome. This approach typically involves several generations of crosses and requires many lines to be discarded, representing wasted effort and resources. Therefore, we sought to create balancer chromosomes that could be directly targeted with transgenes of interest using phiC31-mediated RMCE (Bateman et al. 2006). This approach makes use of a “target cassette,” which consists of a dominant marker gene flanked by attP recognition sites for phiC31 integrase, that is first integrated into the genome. Once the target is established, a “donor cassette” carrying a transgene of interest flanked by phiC31 attB sites can be directly incorporated at the precise genomic position of the target cassette. As described below, our strategy was to use traditional P-element–mediated transgenesis to incorporate dominantly marked RMCE target cassettes onto balancer chromosomes. Once established, these targets can be used to directly incorporate transgenes of interest onto balancer chromosomes.

We previously created a P element that carries an RMCE target cassette consisting of a mini-white gene flanked by phiC31 attP sites (Bateman and Wu 2008). Using a Δ2-3 transposase source, we remobilized existing insertions of this P element and, via three different cross schemes, screened for new insertions onto the X-chromosome balancer FM7h (Heitzler 1997), the second chromosome balancer CyO, and the third chromosome balancer TM3 (see supporting information, File S1 for details of remobilization). For each balancer, we isolated three independent insertions and used inverse PCR and sequence analysis to map the precise genomic positions of the P elements (Table 1). The majority of these insertions mapped to euchromatic regions in or near broadly expressed genes and within chromatin environments expected to facilitate gene expression (Filion et al. 2010; Graveley et al. 2011; Kharchenko et al. 2011) (Table 1, Table S1). One exception, the insertion in line CyOJ01, was mapped to a Doc element that we did not precisely locate.

To assess the potential utility of these lines, we first confirmed that RMCE was supported at appreciable levels using at least one representative target-bearing line for each of the three balancer chromosomes, namely FM7hFS5, CyOJ01, CyOJ08, and TM3FS18. In the presence of a genomic source of the phiC31 integrase (Bischof et al. 2007), we injected donor constructs carrying attB sites flanking either an intronless yellow gene or a fluorescent marker driven by the eye-specific enhancer GMR (Moses and Rubin 1991) (Figure 1 and Table 2). Although we found experimental variation in transformation efficiencies, the lines that we tested supported RMCE at rates up to 41%, consistent with our rates of transgenesis for other genomic targets using this method and our current injection apparatus (data not shown; Bateman and Wu 2008).

Finally, we verified that transgene expression was supported in the transformants that we generated. First, we assessed adult body pigmentation of transformants carrying an insertion of the intronless yellow gene in an otherwise yellow mutant background, and we found that all (9/9) transformed lines produced fully penetrant levels of yellow pigmentation indistinguishable from wild-type flies (Figure 2 and data not shown). To address gene expression at earlier points of development, we assessed the expression of donor cassettes carrying GMR-GFP or GMR-mcherry in whole-mounted late-stage embryos and in eye imaginal discs from wandering third instar larvae (Moses and Rubin 1991). Insertions into FM7hFS5, CyOJ01, CyOJ08, and TM3FS18 produced robust tissue-specific fluorescence (Figure 2, Figure S2), demonstrating that our modified balancers can support gene expression at multiple stages of development.

We anticipate that the modified balancer chromosomes described here will greatly facilitate future efforts at incorporating new transgenes onto balancers and may foster new approaches to balancer chromosome modifications by removing a significant barrier to obtaining balancer insertions. As balancers are developed for other genetic models (Hentges and Justice 2004), a similar scheme for simplified balancer marking may also be beneficial in those systems.


Notes

Communicating editor: K. S. McKim

Acknowledgments

We thank the Bloomington Drosophila Stock Center for fly stocks and for agreeing to distribute the balancer stocks described here; the Mount Desert Island Biological Laboratory DNA Sequencing Core for DNA sequencing; and Michael Palopoli and Bruce Kohorn for helpful comments. This project was supported by grants from the National Center for Research Resources (5-P20-RR-016463-12) and the National Institute of General Medical Sciences (8-P20-GM-103423-12) of the National Institutes of Health.


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Figures

[Figure ID: fig1]
Figure 1 

Injection scheme for RMCE using targets on (A) autosomal or (B) X chromosomal balancers. Germline-targeted phiC31 integrase is supplied from the X-chromosomal ZH2A insertion (Bischof et al. 2007). In the G0 generation, single males or females (A) or females only (B) that carry the RMCE cassette-bearing balancer (50% of progeny expected) are mated singly to flies with a w genotype, and the F1 generation is screened for balanced progeny in which the mini-white eye color of the target cassette is lost. Insertions onto CyO are obtained through a scheme analogous to (A). See Figure S1 for an alternate strategy using stocks carrying the integrase source and the target balancer concurrently.



[Figure ID: fig2]
Figure 2 

Insertion of donor cassettes onto balancers supports expression of transgenes. (A) Schematic of the exchange reaction. Integrase-mediated crossovers at both ends of the aligned target and donor cassettes result in removal of mini-white and integration of the gene of interest (GOI) into the genome. Triangles, att sites; gray boxes, P-element ends. (B) FM7hFS5/Y males before (left) and after (right) RMCE integration of an intronless yellow transgene. The fly on the left retains the mini-white eye pigmentation and lacks yellow pigmentation, whereas on the right, the mini-white eye color is lost, and expression of the yellow transgene is evident in the wing and abdomen. Transgenic insertions of the intronless yellow cassette on CyOJ01 and TM3FS18 produced similar pigmentation (not shown). (C–E) Embryonic expression of GMR-GFP (A, C) or GMR-mCherry (B) insertions onto balancers. White arrowheads, GMR-specific expression (Moses and Rubin 1991); autofluorescence of the gut is also evident.



Tables
[TableWrap ID: t1] Table 1  Balancer chromosomes carrying RMCE target cassettes
Balancer Line Insert Cytology Insert Position Insert Strand Nearest Gene Relative Position of Insertion
FM7hFS2 10B6 X:11266889 Top Dlg1 Genic
FM7hFS4 18A3-4 X:19047768 Top RhoGAP18B Genic
FM7hFS5 6F3 X:6969300 Top Sxl Intergenic
CyOJ01 ND ND ND Doc Element (Repeat sequence)
CyOJ04 37B8 2L:18987255 Top CG10641 Genic
CyOJ08 37F2 2L:19572635 Bottom Spi Genic
TM3FS10 79A2 3L:21872640 Top Mub Genic
TM3FS11 85A5 3R:4502640 Top CG8043 Genic
TM3FS18 100D1 3R:27550495 Bottom ttk Genic

Insertions were mapped by comparing sequences of inverse PCR products to release 5.3 of the Drosophila melanogaster genome sequence. The insertion in the CyOJ01 line was found in a Doc element and was not precisely mapped (ND, not determined). See Table S1 for further information on genome annotations near each insertion.


[TableWrap ID: t2] Table 2  Target cassettes on balancer chromosomes support RMCE
Balancer Line Donor Cassette Gene Embryos Injected Vials with RMCE % Vials with RMCE
FM7hFS5 yellow 175 3/12 25%
FM7hFS5 GMR-GFP 200 1/17 5.9%
TM3FS18 yellow 165 2/13 15%
TM3FS18 GMR-GFP 243 7/17 41%
CyOJ01 yellow 150 4/26 15.4%
CyOJ01 GMR-GFP 150 2/28 7.1%
CyOJ08 GMR-mCherry 250 2/19 10.5%

Donor constructs in sterile water were injected according to the scheme in Figure 1. DNA concentrations were 250 ng/µl (yellow), 325 ng/µl (GMR-GFP), and 115 ng/µl (GMR-mCherry).



Article Categories:
  • Investigations

Keywords: RMCE, targeted transgenesis, phiC31, Drosophila, balancer.

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