Inducible model for beta-six-mediated site-specific recombination in mammalian cells.
The prokaryotic beta recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of beta recombinase (beta-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of beta recombinase (beta-AR) and a triple fusion of beta-EGFP to the same ligand-binding domain (beta-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric beta recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the beta-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus.
Servert
Pilar
P
Departamento de Inmunología y Oncología, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco E-28049, Madrid, Spain.
Garcia-Castro
Javier
J
Díaz
Vicente
V
Lucas
Daniel
D
Gonzalez
Manuel A
MA
Martínez-A
Carlos
C
Bernad
Antonio
A
D000728:Androgens; D000818:Animals; D002460:Cell Line; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D023281:Genomics; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008957:Models, Genetic; D041681:NIH 3T3 Cells; D011944:Receptors, Androgen / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D045522:Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D015533:Transcriptional Activation
D000728:Androgens; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D045522:Recombinases; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase