Recombination Mediated Homolog Exchange (RMHE) is a novel method (Matar & Nonet, 2021) that I developed that permits the manipulation of transgenes integrated at the same locus from being in the trans configuration to being in the cis configuration (Figure 1). The approach is particularly useful to create bipartite driver and reporter combinations in cis to reduce the complexity of segregation of transgenes in crosses.
RMHE is based upon recombination between phiC31 attB and attP sites which are incorporated into transgenes created by RMCE or other similar site specific insertion methods. RMHE permits the combinatorial use of driver and reporters and can even for a single lab provide a robust method for creating high expressing bipartite transgenes.
Currently we are focusing on creating RMHE reagents for insertions at jsSi1579 II, an RMCE landing site 46 bp from the ttTi5605 Mos insertion site. I designed specialized RMCE vectors (pRMHEB and pRMHEP) that incorporate an attB or attP site either 3′ or 5′ of the insert cloning site. These vectors are compatible both with Golden Gate cloning and traditional restriction endonuclease cloning. For example, I cloned the mec-4 promoter driving the tet OFF tetR-L-QF driver into pRMHEB and integrated the clone at jsSi1579 using standard RMCE. In parallel I cloned a tetO 7X ∆mec-7p nls-GFP-C1 reporter into pRMHEP and also integrated that clone at jsSi1579. After outcrossing bqSi711 FLP transgene, the driver transgenes was crossed into jsSi1623 [mex-5p phiC31 sl2 mNeonGreen] as a sources of recombinase, and this heterozygote is crossed into the reporter strain. Reporter/Driver ; phiC31/+ animals are then crossed to wt animals, and Driver attL Reporter/+ recombinants are isolated. The frequency of recombination is robust, with 15% of progeny of a Reporter /Driver animal carrying the cis configuration. The frequency in the male germline is lower with 4% of progeny of male Reporter/Driver animals carrying the cis configuration. However if when the recombinant chromosome can be visually assayed (GFP+) the male frequency is sufficient to easily allow the isolation of the recombinant chromosome either in a male of hermaphrodite offspring.
RMHE is particularly powerful because of the combinatorial manner in which attB and attP lines can be recombined. In the lab we are currently creating a set of ~ 15 distinct promoter driver lines that include most drivers for most C. elegans tissue and about 15 reporter lines including tools for visualizing organelles. This combination will permit the creation of any driver controlling the expression of any reporter by 3 simple crosses. Creating a new reporter or driver compatible with existing line immediate creates the possibility of creating combinations with all existing lines.
In addition, RMHE has the potential to be used in other contexts. In particular, using CRISPR in combination with RMHE will in theory allow one to place any two transgenes previously integrated at the same common mosSCI integration site ( ttTi5605 II, cxTi10882 IV, etc) in cis (Figure 3).
References
Matar & Nonet. (2021). Facile manipulation of bipartite expression transgenes in C. elegans using Recombinase-Mediated Homolog Exchange. unpublished data.
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last updated 8-3-2023