We are developing a more efficient transgene development pipeline called Recombination-Mediated Insertion (RMI) that uses phiC31 recombinase to integrate whole plasmids directly into the genome in single copy at previously integrated phiC31 attB and attP sites. phiC31 RMI uses the combination of phiC31 to integrate the plasmid and FLP to prevent extra-chromosomal array formation (Fig. 1). In addition, further manipulation of the transgene can performed to excise the selection marker by crossing to germline Cre lines (Nonet, 2023). The approach is promising because the efficiency of RMI is over 10 times higher than FLP mediated RMCE (Nonet, 2024). Although the approach was initially developed for integrating into bipartite driver and reporter lines (Fig. 2), we have since developed a landing site on chr IV that contains a self germline recombinase expression cassette that is excised upon integration (Beck and Nonet, 2025).
phiC31 drives the integration and FLP breaks down the array (created initially after injection of DNA) into monomers for integration and to prevent isolating extrachromosomal array transgenes. After integration FLP also excises parts of the integration plasmid and the germline recombinase expression cassette (labeled Tools). If desired, the resistance selection cassette can subsequently be excised by crossing through a germline Cre line. A landing site on Chr II that consists of solely and attP site (flanked by a LoxP and a FRT3) site is also available, but this landing site must be used in conjunction with an unlinked source of phiC31 and FLP (jsSi1784).
The initial implementation of this system was for the creation of linked driver::reporter bipartite expression system transgenes. Over the last several years we have developed numerous tetOFF driver lines and tetO reporter lines that are tagged with a 3′ attB or 5′ attP site, respectively. These strains can be combined by RMHE to create cis-linked driver::reporter transgenes. However, in some cases the appropriate driver or reporter line is not available. RMI provides a versatile and very efficient means of creating new linked bipartite expression lines.
Examples of RMI using phiC31 and FLP which are provided in trans from a single copy transgenic line (not shown). phiC31 drives the integration and FLP breaks down the array (created initially after injection of DNA) into monomers for integration and to prevent isolating extrachromosomal array transgenes. In certain cases, FLP also excises parts of the integration plasmid after phiC31 integration (Figure 2B). If desired, the Neo resistance selection cassette can be excised by crossing through a germline Cre line. Shown are examples integrating into unmarked driver and reporter lines. However, the same approach can also be used to integrate into cis-marked lines created using rRMCE (Nonet, 2023).
A) RMI of an attB driver plasmid into an attP marked tetO reporter line.
B) RMI of an attP reporter plasmid into an attB marked tetOFF driver line. Note that FLP also excises the AmpR and attR site from the unstable intermediate after integration by phiC31.
The currently developed system depends on phiC31 recombinase and FLP recombinase provided in trans. As of Dec. 2023, jsSi1784 IV is the only transgene that permits performing RMI. Currently, any attB site integrated in the genome can be used in combination with jsSi1784 and the vector pAttPF3Neo to create transgenes using either traditional ligation or Golden Gate cloning to create in targeting plasmid. Successful injection of a single animal is all that is required to get an insertion. Indeed, if one co-injects multiple plasmids one can isolate up to a dozen distinct plasmids inserted from the injection of a single animal though not all independent insertions occur at the same frequency (Nonet, in preparation).
RMI reagents
References
- Nonet, M.L. (2023). Rapid generation of C. elegans single copy transgenes combining RMCE and drug selection. Genetics iyad072. PMID:37079426 [Abstract] [PDF]
- Nonet, M.L. (2024) Creation and manipulation of bipartite expression transgenes in C. elegans using phiC31 recombinase. bioRxiv DOI:10.1101/2024.03.01.583017
- Beck, A.A. and Nonet, M.L. A landing site for efficient single step transgenesis using recombination mediated insertion, in preparation as micropublication
Last Edited: 6/14/2025 by MLN