MN: Conceptualization, Funding acquisition, Methodology, Formal analysis, Writing - original draft, Writing - review and editing
Recombination-mediated cassette exchange (RMCE) is a recently developed alternative method for creating single copy transgenes using recombination rather than repair of double stranded breaks as the mechanism for driving integration into the genome. Two alternative methods for performing RMCE have been developed; a two-component approach using an unlinked source of FLP recombinase, and a one-component approach using a FLP expression cassette within the landing site. Here, I describe new landing sites for performing both types of RMCE. The new landing sites are located within 50 bp of well-vetted MosSCI insertion sites on Chr II and Chr IV.
A) Structure of the
B) Position and structure of the genomic interval of landing sites.
C) Comparison of the expression levels of identical insertions at various RMCE landing sites. Expression level of a
D) Images of the gland cell background GFP expression of transgenic animals carrying
E) Frequency of insertions obtained at distinct landing sites. All injected animals were counted regardless of perceived quality of injection or survival. In most cases only a single gonad was injected. F1 Rol progeny were typically grouped 6 per plate for identification of integrated lines. Lines represents the number of independent F1 progeny plates that segregated an integrated line. Success is defined as obtaining an integration event at the expected genomic position. The insert size does not include the 7.85 kb vector and SEC sequences excised during heat shock. § Injections performed using PB washed DNA.
Transgenic animals are powerful tools in the study of basic biological processes using
The RMCE approach I developed takes advantage of two distinct recombinases. A plasmid template delivered into the gonad of young adult animals is first integrated into the genome using FLP recombinase. Both the template and the landing site contain two distinct FLP integration sites,
Two methods have been developed for performing RMCE. The first method uses a landing site and an unlinked source of FLP recombinase (usually
I integrated a two-component landing site using CRISPR/cas9 just adjacent to the position of the
Recently developed bipartite reporters including a
In developing a new recombination-mediated homolog exchange technique (
I previously demonstrated that RMCE yields insertions of expected structure in greater than 95% of cases (Nonet, 2020). I have also characterized most of the transgenes obtained at these additional landing sites either by confirming the presence of an expected fluorescence pattern, recombinase activity,
The new landing site are now available at the CGC and should provide additional flexibility in creating RMCE-based transgenic animals. I also plan to create additional landing sites at well-characterized high expressing genomic positions on the remaining chromosomes that currently do not contain landing sites.
Inserts were cloned into pLF3FShC (Nonet, 2020), pRMHEB or pRMHEP (
For quantification of GFP signals, homozygous L4 hermaphrodite animals were mounted on 2% agar pads in a 2 µl drop of 1mM levamisole in phosphate buffered saline, cover slipped and imaged on an Olympus (Center Valley, PA) BX-60 microscope equipped with a Qimaging (Surrey, BC Canada) Retiga EXi monochrome CCD camera, a Lumencor AURA LED light source, Semrock (Rochester, NY) GFP-3035B and mCherry-A-000 filter sets, and a Tofra (Palo Alto, CA) focus drive, run using micro-manager 2.0ß software (Schindelin
Integration vectors were assembled using Golden Gate (GG) reactions as described in Nonet (2020). Other plasmids were constructed using standard cloning techniques.
The following previously published plasmids were used:
pBluescript KS (+) (Short
The following plasmids were constructed:
NMp3143 peft-3-cas9 (3 int)
A derivative of the pDD162 Cas9 expression plasmid lacking the empty U6 sgRNA cassette. pDD162 was amplified using NMo5228/5379 and re-circularized using a Gibson assembly reaction.
NMp3150 DR274 U6 FE
U6 promoter sgRNA clone with a flipped and extend sgRNA as described in Ward (2015). sgFE was amplified from NMp3055 using NMo5407/5075, purified, digested with BamHI and HindIII, and inserted into BamHI and HindIII digested NMp3055.
NMp3153 DR274 U6 FE dpy-10
U6 promoter sgRNA targeting
NMp3630 DR274 U6 MosII
U6 promoter sgRNA targeting Chr II adjacent to the
NMp3828 DR274 U6 sgTi1453 F
U6 promoter sgRNA targeting Chr I adjacent to the
NMp3829 DR274 U6 sgMosL
U6 promoter sgRNA targeting
NMp4043 pSAP ChrII FLP loxP FRT FRT3 landing
Chr II full RMCE landing site CRISPR template. The left arm amplified from N2 genomic DNA using NMo6707/6450, the right arm amplified from N2 genomic DNA using NM06708/6453, and the loxP FLP FRT FRT landing site from NMp3746 were co assembled into NMp3421 using a SapI Golden Gate assembly reaction.
NMp4053 DR274 5′ arm cxTi10882 left arm
Left arm genomic fragment adjacent to
NMp4054 DR274 3′ arm cxTi10882 right arm
Right arm genomic fragment adjacent to
NMp4055 DR274 U6 cxTi10882
U6 promoter sgRNA targeting Chr IV adjacent to the
NMp4057 pSAP cxTi10882 FLP loxP FRT FRT3 landing
Chr IV full RMCE landing site CRISPR template. The left chr IV arm from NMp4053, the right Chr IV arm from NMp4054 and the loxP FLP FRT FRT landing site from NMp3746 were co assembled into NMp3421 using a SapI Golden Gate assembly reaction.
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3887 | ACCGGAAACCAAAGGACGAGAG |
3888 | ACGCCCAGGAGAACACGTTAG |
3889 | CCAAACAAGTGTCGTTGACCCAG |
3890 | CATATCCGCCAAGGACGCTC |
5075 | GCCAAGCTTCACAGCCGACTATGTTTGGCGTC |
5228 | CGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG |
5236 | TTTGCTACCATAGGCACCACGAG |
5237 | AAACCTCGTGGTGCCTATGGTAG |
5238 | ACTTGAACTTCAATACGGCAAGATGAGAATGACTGGAAACCGTACCGCATGCGGTGCCTATGGTAGCGGAGCTTCACATGGCTTCAGACCAACAGCCTAT |
5379 | CGTCGTGACTGGGAAAACCCTGGCGTTCCCAACAGTTGCGCAGCC |
5407 | AAGGATCCGGGTCTCAGTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCG |
6439 | TTTGATATCAGTCTGTTTCGTAA |
6440 | AAACTTACGAAACAGACTGATAT |
6450 | TTGCTCTTCATGGCCTCTGAACTGGTACCTC |
6453 | TTGCTCTTCATACCTTGCCATTGTTTCCTG |
6564 | CATCCCATTCACGGCACAAC |
6569 | AAGCTCTTCACTCCGCATTTTCTCCCACCCTG |
6707 | AAGCTCTTCACGCGAAACAGACTGATATCGAAAC |
6708 | AAGCTCTTCAACGTAACGGTCTTCTGTATAAC |
6757 | TTTGATTCACGGCACAACATACAT |
6758 | AAACATGTATGTTGTGCCGTGAAT |
6759 | TTTGATTCACGGCACAACATACAT |
6760 | AAACATGTATGTTGTGCCGTGAAT |
6761 | AACAATTCATCCCATTCACGGCACAACATATGGCGGCCGCTCTAGAACTAGGCTGTTTCG |
7058 | AGGTCTCAGACGCTGTGTAGCGGTCCTCTATTG |
7059 | AGGTCTCTCTACAGTCGCATACGTCGTATCCC |
7060 | GGTCTCAGTGGTTTCAACGGTGGAAGAAGGG |
7061 | CGGTCTCTTCGCACAGGCATCCAACAGTACG |
7062 | TTTGACTGTTGGATGCCTGTGTAG |
7063 | AAACCTACACAGGCATCCAACAGT |
 
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Chr I landing site |
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This study |
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Chr
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This study |
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Chr IV landing site |
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This study |
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Chr II landing site |
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This study |
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7X tetO GFP-C1 |
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This study. RMCE insertion of NMp3774 into
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This study. RMCE insertion of NMp3732 into
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This study. RMCE insertion of NMp3732 into
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This study. RMCE insertion of NMp3732 into
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BN711 |
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Macías-León and
Askjaer (2018); CGC |
EG8992 |
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Schwartz
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NM5196 |
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Nonet (2020); CGC |
NM5209 |
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Nonet (2020) |
NM5228 |
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Nonet (2020); CGC |
NM5236 |
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Nonet (2020) |
NM5264 |
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Nonet (2020) |
NM5295 |
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Nonet (2020) |
NM5322 |
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This study |
NM5327 |
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This study |
NM5337 |
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This study |
NM5402 |
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This study |
NM5467 |
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Nonet (2021) |
NM5471 |
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This study |
NM5500 |
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This study |
NM5580 |
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This study |
NM5582 |
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This study |
NM5633 |
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This study |
Plasmids are available by request from MLN. Strains containing the four new landing sites have been submitted to the
WUMS Department of Neuroscience funds and R01 GM141688.