Untitled

Abstract

We constructed and tested a Cre-loxP recombination-mediated vector system termed pCrox for use in transgenic plants. In this system, treatment of Arabidopsis under inducing conditions mediates an excision event that removes an intervening piece of DNA between a promoter and the gene to be expressed. The system developed here uses a heat-shock-inducible Cre to excise a DNA fragment flanked by lox sites, thereby generating a constitutive GUS reporter gene under control of the CaMV 35S promoter. Heat-shock-mediated excision of several, independent lines resulted in varying degrees of recombination-mediated GUS activation. Induction was shown to be possible at essentially any stage of plant growth. This single vector system circumvents the need for genetic crosses required by other, dual recombinase vector systems. The pCrox system may prove particularly useful in instances where transgene over-expression, or under-expression by antisense, would otherwise affect embryo, seed or seedling viability.

Generation of marker free salt tolerant transgenic plants of Arabidopsis thaliana using the gly I gene and cre gene under inducible promoters

“…This resulted in the construct pnpt-lox + rd29A-glyI, wherein the gene of interest, glyI was placed under the control of the rd29A promoter. Finally, a DNA fragment comprising the cre-recombinase (hsp-cre) driven by the heat-shock inducible promoter was obtained as a PCR product using primers with EcoRI and BamHI overhangs (5 0 -GCCAGAATTCATCGGTTTGAAGATG GCAAGTGTT-3 0 and 5 0 -AATTGGATCCTAATCG CCATCTTCCAGCA-3 0 ) and the pCrox 18 vector (Hoff et al 2001) as the template. The CaMV35S terminator was obtained as a BamHI-EcoRI digest from dsProA (Pooggin et al 2003).…”

“…The hsp-cre fragment from the pCrox18 vector (Hoff et al 2001) was also introduced in the same vector within the loxP sites (see Materials and methods and Fig. 1).…”

“…Recently, various approaches were investigated to reproducibly obtain optimum CRE activity (Marjanac et al 2008). An exciting area that deserves attention is the use of site-specific recombinases under the control of inducible promoters to excise SMGs after transgenic plants have been recovered (Hoff et al 2001;Cuellar et al 2006). Based on this premise, the cre gene under a heat shock inducible promoter (hsp) was cloned in a binary vector containing the glyI gene so that the cre gene, the loxP sequences and the glyI gene can be transferred to the target species.…”

Elimination of Transgenic Sequences in Plants by Cre Gene Expression

“…The heat-shock regulated system seems to be the simplest and most familiar for use. Its function has been demonstrated as functional in Arabidopsis (Hoff et al, 2001), tobacco , potato (Cuellar et al, 2006), maize (Zhang et al, 2003), rice (Khattri et al, 2011) and aspen (Fladung and Becker, 2010). In the chemically regulated self-excision system developed by Zuo and associates (2001), the cre gene was combined with the XVE system which is induced by β-estradiol.…”

“…The essential feature of conditional Cre systems is a careful regulation with respect to time and tissue. Background Cre activation was observed for heat-shock inducible (Hoff et al, 2001;Wang et al, 2005) and some seed-specific promoters (Odell et al, 1994;Moravčíková, et al, 2008), resulting in reduced efficiency of the systems.…”