Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana

Abstract

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable. We have evaluated the potential of seven different germline promoter:cre fusions to remove a stably integrated lox cassette through Cre-mediated recombination in Arabidopsis thaliana. We monitored the functionality of each promoter in the germline of primary transformants by analyzing the presence of the recombined lox cassette in T(2) progeny. The selected germline promoters are involved in different developmental cues, including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1). For five out of these seven promoters we were able to show that efficient Cre-mediated recombination does, indeed, occur and that the recombination takes place at some point during germline development. Furthermore, a recombination efficiency of 100% is obtained when Cre-expression is regulated by the CLAVATA3 promoter. In addition, with these promoters, we observe much less variation in recombination frequency than previously reported for the 35S promoter. For these reasons, we believe that germline-specific Cre-lox recombination provides an additional tool to the site-specific recombination technology in plants.

Less is more: strategies to remove marker genes from transgenic plants

“…Verweire et al also successfully demonstrated the use of Arabidopsis germline-specific promoters from the APETALA1 and SOLO DANCERS genes to achieve genetically programmed, Cre-recombinase-mediated auto-excision for SMG-free plants [92]. More recently, Frédéric et al evaluated seven different germline-related promoters for their suitability in regulating Cre expression in transgenic Arabidopsis [93] . Five out of the seven promoters, which varied in developmental stages and tissues were able to drive efficient Cre-mediated gene excision.…”

Employing Site-Specific Recombination for Conditional Genetic Analysis in Sinorhizobium meliloti

“…The enzymes Cre and Flp are members of the tyrosine recombinase family that catalyze crossovers between the 34-bp recognition sites loxP and FRT, respectively (14,25). The Cre/loxP and Flp/FRT systems have been successfully used to cause programmed gene deletions and genome rearrangements in various model organisms (2,5,29,33), but their use for conditional genetics in bacteria has been limited (19).…”

Transposon tagging in diploid strawberry

“…, 1999) revealed that both of the nearest polyA signal motifs would have been separated from the gene by T‐DNA insertion, reinforcing the suggestion of continued transcription into the T‐DNA. The successful generation of unique transposants by LP7, by its fortuitous occurrence in a gametophyte‐specific gene, supports the utilization of gametophyte‐ or meiosis‐specific promoters for driving TPase expression for germ‐line transposon tagging systems (Van Ex et al. , 2009).…”