Development of a chromosomally integrated metabolite-inducible Leu3p-alpha-IPM "off-on" gene switch.
BACKGROUND
Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression.


RESULTS
Here, we show that a chromosomally integrated yeast 'Leu3p-alpha-IotaRhoMu' system constitutes a ligand-inducible regulatory "off-on" genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of alpha-isopropylmalate (alpha-IotaRhoMu) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable alpha-IPM presents an EC(50) equal to 0.8837 mM and fast "OFF-ON" kinetics (t(50)ON = 43 min, t(50)OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo.


CONCLUSIONS
Our results demonstrate that the 'Leu3p-alpha-IotaRhoMu' constitutes a simpler and safer system for inducible gene expression in biomedical applications.
Poulou
Maria
M
Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Center "Alexander Fleming," Attica, Greece.
Bell
Donald
D
Bozonelos
Kostas
K
Alexiou
Maria
M
Gavalas
Anthony
A
Lovell-Badge
Robin
R
Remboutsika
Eumorphia
E
D000818:Animals; D001483:Base Sequence; D033481:Chromosomes, Mammalian / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D008293:Malates / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D011247:Pregnancy; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
C063667:LEU3 protein, S cerevisiae; D008293:Malates; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C005906:alpha-isopropylmalate
Mol Cell Biol. 1994 Aug;14(8):5547-57
8035829
Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9209-14
11481484
Neurochem Res. 1999 Sep;24(9):1181-8
10485590
Microbiol Mol Biol Rev. 2003 Mar;67(1):1-15, table of contents
12626680
Methods Mol Biol. 2008;420:97-117
18641943
J Clin Invest. 1997 Dec 1;100(11):2865-72
9389753
Curr Biol. 1998 Dec 3;8(24):1323-6
9843687
J Antibiot (Tokyo). 2001 Jan;54(1):44-55
11269714
Nat Genet. 2000 Apr;24(4):372-6
10742100
Curr Genet. 1983 Sep;7(5):369-77
24173418
Mol Cell Biol. 1993 Sep;13(9):5702-9
8355711
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3346-51
8622939
Methods. 1998 Apr;14(4):381-92
9608509
J Embryol Exp Morphol. 1977 Feb;37(1):65-77
558277
J Clin Invest. 2007 Jul;117(7):1893-901
17607363
Handb Exp Pharmacol. 2007;(178):49-72
17203651
Lab Invest. 2006 Jul;86(7):664-75
16652107
FEBS Lett. 1996 Jul 22;390(2):191-5
8706857
Nucleic Acids Res. 2001 May 15;29(10):E47
11353092
Pharmacology. 2003 Oct;69(2):74-8
12928580
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10933-8
8855286
EMBO Rep. 2006 May;7(5):496-9
16670683
J Biol Chem. 1999 Jul 2;274(27):19017-24
10383402
Nucleic Acids Res. 1998 Jul 1;26(13):3263-9
9628928
Mol Ther. 2002 Aug;6(2):238-42
12161190
Dev Biol. 2009 Jan 1;325(1):171-8
19013447
Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51
1319065
Exp Cell Res. 2000 Nov 1;260(2):334-9
11035928
Science. 1992 Nov 13;258(5085):1143-5
1439822
J Am Soc Nephrol. 2000 Nov;11 Suppl 16:S95-S100
11065338
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10887-90
8855277
Cell. 2001 Mar 9;104(5):675-86
11257222