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. 2005 Sep 28;127(38):13088-9.
doi: 10.1021/ja0531226.

Light activated recombination

Kristian H Link  1 , Youheng Shi, John T Koh
Affiliations

Light activated recombination

Kristian H Link et al. J Am Chem Soc. .

Abstract

Many genes elicit their actions through their expression in precise spatial patterns in tissues. Photoregulated expression systems offer a means to remotely pattern gene expression in tissues. Using currently available photopatterning methods, gene expression is only transient. Herein is described a general method to permanently alter a cell's genome under the control of light. The photocaged estrogen receptor (ER) antagonists, nitroveratryl-hydroxytamoxifen (Nv-HTam) and nitroveratryl-hydroxytamoxifen aziridine (Nv-HTaz), mediate exposure-dependent recombination in cells expressing the Cre-ER, a fusion of the site-specific recombinase Cre and ER. Both Nv-HTam and Nv-HTaz only activate recombination by Cre-ER after exposure to light. When released only intracellularly, the covalent-modifying Taz can mediate significant amounts of recombination in an exposure-dependent manner. Nv-HTaz and Cre-ER represent perhaps the first compound that can be used to photopattern gene expression through recombination.

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Figures

FIGURE 1
FIGURE 1
(A). Photocaged hydroxytamoxifen (HTam). (B). Taz forms a covalent adduct with Cys 530 of ER; Nv-HTaz is a photocaged Taz analog. (C). Cre-ERT can be used to permanently turn on reporter gene (LacZ) expression (and turn off Neor) through recombination. (HSP; heat shock protein, Neor; neomycin resistance).
FIGURE 2
FIGURE 2
Nv-HTam can regulate recombination dependent β-gal expression (x-gal stain) in HEK293 cells expressing pLacZ(LNL) and pCre-ERT/Zeo. (A) No ligand, unexposed. (B) No ligand, exposed (180s×3W). (c) Nv-HTam, unexposed. (D) Nv-HTam, exposed. Intracellular and extacellular ligand (200 nM) with 200 nM trans-retinoic acid (tRA).
FIGURE 3
FIGURE 3
Comparison of Nv-HTam and Nv-HTaz mediated recombination after 72 h (x-gal staining). (A) Nv-HTaz, unexposed. (B) Intra- and extra-cellular uncaged Nv-HTaz, exposed (180s) (C) Intracellularly uncaged Nv-HTam, exposed (180s). (D) Intracellularly uncaged Nv-HTaz, exposed (180s). (E) Intracellularly uncaged Nv-HTam, exposed 3-times (45s). (F) Intracellularly uncaged Nv-HTaz, exposed 3 times (45s). 1000 nM ligand plus of 200 nM tRA.
FIGURE 4
FIGURE 4
β-gal expression measured by OPNG assay for cells treated with Nv-HTaz or Nv-HTam with 200 nM tRA and exposed three times at 24 h intervals (45 s×3W).(p; unpaired t-test)
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