	US 7,348,179 B2
	IRES enabled gene trapping in plants
Yuri Gleba, München (Germany); Newell Bascomb, Wayne, N.J. (US); Mark Bossie, Robbinsville, N.J. (US); Gerald Hall, Jr., Morrisville, Pa. (US); and Thomas J. Petty, II, Ewing, N.J. (US)
Assigned to Icon Genetics, Inc., Princeton, N.J. (US)
Appl. No. 10/343,498 PCT Filed Apr. 17, 2002, PCT No. PCT/US02/11924 § 371(c)(1), (2), (4) Date Jun. 17, 2003, PCT Pub. No. WO02/083867, PCT Pub. Date Oct. 24, 2002.
Claims priority of provisional application 60/284239, filed on Apr. 17, 2001.
Prior Publication US 2004/0014216 A1, Jan. 22, 2004
Int. Cl. C12N 15/82 (2006.01); C12N 5/14 (2006.01); C12N 5/04 (2006.01); A01H 5/00 (2006.01)

U.S. Cl. 435—468 [435/419; 435/320.1; 800/278; 800/295]

12 Claims

1. A method of introducing a structural gene of interest into plants, comprising:

(a) providing a plant cell having in its transcribed region a first nucleic acid construct comprising in operable association, at least one tobamovirus IRES (internal ribosome entry site), at least one site-specific recombination site and a reporter gene;

(b) providing the plant cell of (a) with a site-specific recombinase which recognizes said site-specific recombination site(s) wherein said recombinase is provided recombinantly, and wherein recombinase-recombination site is selected from the group consisting of CRE-lox from bacteriophage PI, FLP-FRT from Saccharomyces cerevisiae, R-RS from Zygosaccharomyces rouxii, Gin-gix from bacteriophage Mu, integrase/att from bacteriophage Phi C31, and yeast endonuclease I-Scel;

(c) introducing into the plant cell of (a) a second nucleic acid construct comprising a structural gene of interest flanked by recombination sites such that said structural gene of interest is integrated into the first nucleic acid construct at the site-specific recombination site(s) and wherein said structural gene of interest is under operable control of the IRES, wherein said site-specific recombinase catalyzes integration of the structural gene into the first nucleic acid construct; and

(d) selecting for plant cells having the structural gene of interest integrated into the first nucleic acid construct, and which is under operable control of the IRES.