Cre/loxP Recombination-Mediated Cassette Exchange (RMCE) in Drosophila

RMCE was designed to transfer transgenes into the confined genomic locations. There are currently several similar systems available. The system we are introducing here uses Cre-mediated recombination to insert transgenes into precise genomic "landing sites" (Fig. 1). By eliminating potential "position effects" from nearby endogenous regulatory elements, precise comparisons between multiple constructs can be made. Please refer to service page for details.

Fig. 1. A typical Cre-loxP RMCE reaction. Two separate Cre recombination events cause the precise exchange of donor construct and landing site sequence. Donor construct contain multiple cloning sites (MCS) for inserting transgene fragments.

The processes are as follow:
1. Microinject the donor construct and the helper Cre recombinase into "landing site" line(s)
2. Cross surviving flies to yw flies
3. Score offspring for exchange event (loss of y and gain of w)
4. (optional) PCR confirmation of cassette exchange

The "Landing site" lines currently available at BestGene are:

Line	Location1	Landing site	Expression level2	Transformation efficiency3
X388	X-?	loxP-y-lox2272	?	?
X393	X-?	loxP-y-lox2272	?	?
A11	2L(34B), 13399006	loxP-y-lox2272	++	++
A10	2L(37), 19572636	loxP-y-lox2272	+++	+
A8	3L(68A), 11171208	loxP-y-lox2272	+++4	+++
A13	3R(96F), 21566290	loxP-y-lox2272	++	+

¹The cassette located before the nucleotide number, e.g. A8: inserted before nucleotide 11171208 on chromosome 3L
²Expression level was measured by beta-gal assay
³Transformation efficiency was tested at the Small lab and at BestGene Inc.
⁴A8 has a slight position effect - staining at the anterior tip of the embryo; A11/A10/A13 has no obvious position effect.
(X388 and X393 are not open for ordering now)

Please contact Prof. Small for the vector plasmid.

Prof. Stephen Small
Associate Professor of Biology
New York University
Department of Biology
1009 Silver Building
100 Washington Square East
New York, NY 10012
U.S.A.

Tel: +1-212-998-8244
Fax: +1-212-995-4015