Self-excision of the antibiotic resistance gene nptII using a heat inducible Cre-loxP system from transgenic potato

Abstract

Resistance to antibiotics mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, regulatory agencies and consumer concerns favor these to be eliminated from food crops. Several excision systems exist but none have been optimized or shown to be functional for clonally propagated crops. The excision of the nptII gene conferring resistance to kanamycin has been achieved here using a gene construct based on a heat-inducible cre gene producing a recombinase that eliminates cre and nptII genes flanked by two loxP sites. First-generation regenerants with the Cre-loxP system were obtained by selection on kanamycin media. Following a heat treatment, second generation regenerants were screened for excision by PCR using nptII, cre, and T-DNA borders primers. Excision efficiency appeared to be at 4.7% depending on the heat treatment. The footprint of the excision was shown by sequencing between T-DNA borders to correspond to a perfect recombination event. Selectable marker-free sprouts were also obtained from tubers of transgenic events when submitted to similar heat treatment at 4% frequency. Spontaneous excision was not observed out of 196 regenerants from untreated transgenic explants. Biosafety concerns are minimized because the expression of cre gene driven by the hsp70 promoter of Drosophila melanogaster was remarkably low even under heat activation and no functional loxP site were found in published Solanum sequence database. A new plant transformation vector pCIP54/55 was developed including a multiple cloning site and the self-excision system which should be a useful tool not only for marker genes in potato but for any gene or sequence removal in any plant.

Marker-Free Transgenic Plants through Genetically Programmed Auto-Excision

“…Recently, the company Renessen received U.S. regulatory approval (http://www.aphis.usda.gov/ brs/aphisdocs2 /04_22901p_com.pdf) for the transgenic line LY038 from which the selectable marker, originally present between tandemly oriented lox sites, was removed through introduction of the cre gene by a sexual cross (Ow, 2007). In the auto-excision strategy, activation of the recombinase can be induced either chemically (Sugita et al, 2000;Zuo et al, 2001;Schaart et al, 2004;Sreekala et al, 2005;Zhang et al, 2006) or by heat shock (Kilby et al, 1995;Hoff et al, 2001;Zhang et al, 2003;Wang et al, 2005;Cuellar et al, 2006). In the available strategies, a lot of extra work has to be invested to obtain marker-free homozygous plants.…”

Generating Marker-Free Transgenic Wheat Using Minimal Gene Cassette and Cold-Inducible Cre/Lox System

“…The efficiency of this system differed from our results. The lowest level has been detected in potato shoot internodes (5-14%) (Cuellar et al 2006), Wang et al (2005) have reached 70-80% Cre/lox activity in tobacco seed and leaf, while the excision rate has even reached 100% in tobacco seedlings (Liu et al 2005). Heat shock can cause damage in wheat tissues therefore it is not the most proper way of inducing gene expression.…”

Generation of Transgenic Rice without Antibiotic Selection Marker through Agrobacterium-mediated Co-transformation System

“…The Cre-loxP system was developed to remove target gene by cleavage of site-specific DNA fragment from genomic DNA [7]. However, this approach requires another insertion of cre gene and there is concern about another recombination.…”