Cre-lox Site-specific Recombination System Research Articles

Gene Stacking in Plants Through the Application of Site-Specific Recombination and Nuclease Activity.

Biotechnology methods for inserting genes one by one or as a block of fragment into plant genomes are needed to introduce valuable traits into crop varieties. Insertion of multiple genes into a single site, called as molecular stacking, is important to allow co-inheritance of the genes into the progeny. Generally, two approaches are available for creating gene stacks: nuclease-induced targeted gene integration into native sites and recombinase-mediated gene integration into the engineered sites. The recombinase application is attractive as several recombinases show high efficiency and precision in plant genomes. This chapter describes a gene stacking method based on the use of Cre-lox site-specific recombination system to integrate genes into the engineered sites and nucleases to delete selection genes leading to stacking of traits into a single genomic site. High efficiency and precision, and undetectable off-target effects of Cre-lox in a number of plant species, make it an attractive tool for complex applications such as gene stacking.

Coupling the recombineering to Cre-lox system enables simplified large-scale genome deletion in Lactobacillus casei

BackgroundLactobacillus casei is widely used in the dairy and pharmaceutical industries and a promising candidate for use as cell factories. Recently, genome sequencing and functional genomics provide the possibility for reducing L. casei genome. However, it was still limited by the inefficient and laborious genome deletion methods.ResultsHere, we proposed a genome minimization strategy based on LCABL_13040-50-60 recombineering and Cre-lox site-specific recombination system in L. casei. The LCABL_13040-50-60 recombineering system was used to introduce two lox sites (lox66 and lox71) into 5′ and 3′ ends of the targeted region. Subsequently, the targeted region was excised by Cre recombinase. The robustness of the strategy was demonstrated by single-deletion of a nonessential ~ 39.3 kb or an important ~ 12.8 kb region and simultaneous deletion of two non-continuous genome regions (5.2 and 6.6 kb) with 100% efficiency. Furthermore, a cyclical application of this strategy generated a double-deletion mutant of which 1.68% of the chromosome was sequentially excised. Moreover, biological features (including growth rate, electroporation efficiency, cell morphology or heterologous protein productivity) of these mutants were characterized.ConclusionsTo our knowledge, this strategy is the first instance of sequential deletion of large-scale genome regions in L. casei. We expected this efficient and inexpensive tool can help for rapid genome streamlining and generation restructured L. casei strains used as cell factories.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

The plastid clpP1 protease gene is essential for plant development.

Plastids of higher plants are semi-autonomous cellular organelles that have their own genome and transcription-translation machinery. Examples of plastid functions are photosynthesis and biosynthesis of starch, amino acids, lipids and pigments. Plastid functions are encoded in approximately 120 plastid genes and approximately 3,000 nuclear genes. Although many embryo and seedling lethal nuclear genes are required for chloroplast biogenesis, until now deletion of plastid genes either had no phenotypic consequence (8 genes), or caused a mutant phenotype but did not affect viability (13 genes). Here we identify an essential plastid gene. By using the CRE-lox site-specific recombination system we have deleted clpP1 (caseinolytic protease P1), one of the three genes (clpP1, ycf1 and ycf2) whose disruption had previously only been possible in a fraction of the 1,000-10,000 plastid genome copies in a cell. Loss of the clpP1 gene product, the ClpP1 protease subunit, results in ablation of the shoot system of tobacco plants, suggesting that ClpP1-mediated protein degradation is essential for shoot development.

The presence of a chromatin boundary appears to shield a transgene in tobacco from RNA silencing.

We present isogenic transgenic tobacco lines that carry at a given chromosomal position a beta-glucuronidase (GUS) reporter gene either with or without the presence of the matrix-associated region known as the chicken lysozyme A element. Plants were generated with the Cre-lox site-specific recombination system using heterospecific lox sites. Analysis of GUS gene expression in plant populations demonstrates that the presence of the A element can shield against RNA silencing of the GUS gene. Protection was observed in two of three independent tobacco transformants. Plants carrying an A element 5' of the GUS gene always had stable GUS activity, but upon removal of this A element, the GUS gene became silenced over time in two lines, notably when homozygous.

Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system.

Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids. However, once transformation is accomplished, having the marker gene becomes undesirable. Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome. The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>). Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen. Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene. However, the large deletions were absent when cre was introduced by pollination. Thus pollination is our preferred protocol for the introduction of cre. Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation. The nuclear cre gene could subsequently be removed by segregation in the seed progeny. The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

A Ti-plasmid-convertible .LAMBDA. Phage Vector System Harboring the Hygromycin B Phosphotransferase Gene.

We constructed new λTI phage vectors, λTI3, λTI4, λTI5 and λTI6, which were automatically convertible into Ti-plasmid binary vectors by using the Cre-lox site-specific recombination system of bacteriophage P1. Hygromycin B phosphotransferase (HPT) gene was inserted in the T-DNA region of these λTI vectors as a plant selectable maker. Tobacco plants, which were transformed by Agrobacterium containing the Tiplasmids resulted from the automatic conversion of the λTI vectors, could be tightly selected by hygromycin B. This λTI system will certainly provide a powerful tool for genetic complementation test of candidate genes obtained by positional cloning.

Single-site manipulation of tomato chromosomes in vitro and in vivo using Cre-lox site-specific recombination.

With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 bp specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.

A system for insertional mutagenesis and chromosomal rearrangement using the Ds transposon and Cre—lox

A system for insertional mutagenesis and chromosomal rearrangement in Arabidopsis has been developed. The T-DNA vectors are based on the maize transposon Ds, lox sites from the Cre-lox site-specific recombination system, and transcriptional fusions expressing Ac transposase or Cre recombinase. The engineered transposon is termed Dslox. Transposed Dslox insertions were created by crossing plants bearing Dslox with plants expressing Ac transposase, then simultaneously selecting for excision and reinsertion in F2 seedlings using the herbicides chlorsulfuron and phosphonothricin, respectively. F2 plants bearing stable Dslox insertions were identified by scoring for the absence of the Ac transposase T-DNA, using a novel, visual marker in that T-DNA. Two independent Dslox insertions were characterized and placed 5.6 and 16.5 cM from their T-DNAlox, which mapped close to m506 on chromosome 4. Plants bearing either of the two different transposed Dsloxs and T-DNAlox were crossed to plants expressing Cre recombinase, which catalyzed recombination between the lox site in transposed Dslox and the lox site in T-DNAlox. Lox-lox recombinants were identified selectively amongst progeny of these crosses. Molecular and genetic analysis of the lox-lox rearrangements indicated that both were inversions. The smaller inversion was germinally transmitted from generation to generation as a simple trait, whereas the larger inversion was not transmitted to progeny of plants bearing the rearrangement.

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome.

The bacteriophage P1 Cre-lox site-specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild-type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild-type and mutant lox sites were used in conjunction with two strategies for abolishing post-integration Cre activity: (i) promoter displacement of a cre-expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild-type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild-type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre-lox site-specific recombination system is useful for the single-copy integration of DNA into a chromosomal lox site.

Site-specific cleavage of chromosomes in vitro through Cre-lox recombination.

Site-specific recombination systems are useful tools for chromosome engineering in vivo and site-specific DNA cleavage methods have applications in genome analysis and gene isolation. Here, we report a new method to fragment chromosomes in vitro using the Cre-lox site-specific recombination system. Two lox sites were targeted into the 5.7 Mb chromosomes I of Schizosaccharomyces pombe. In vitro recombination between chromosomal lox sites and exogenously provided lox oligonucleotides 'cleaved' the chromosome at the defined lox sequences. Site-specific cleavage of lox sites in the tobacco genome was also demonstrated. This recombination-based cleavage method provides a novel approach for structural and functional analyses of eukaryotic chromosomes as it allows direct isolation of chromosome regions that correspond to phenotypes revealed through Cre-lox mediated chromosome rearrangements in vivo. Moreover, recombination with end-labeled lox oligonucleotides would permit the specific end-labeling of chromosome segments to facilitate the long range mapping of chromosomes.

Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.

Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system.

The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.

Lambda YES: a multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations.

This work describes a multifunctional phage lambda expression vector system, lambda YES, designed to facilitate gene isolation from eukaryotes by complementation of Escherichia coli and Saccharomyces cerevisiae mutations. lambda YES vectors have a selection for cDNA inserts using an oligo adaptor strategy and are capable of expressing genes in both E. coli and S. cerevisiae. They also allow conversion from phage lambda to plasmid clones by using the cre-lox site-specific recombination system, referred to here as automatic subcloning. A simple method has been developed for the conversion of any plasmid into a phage lambda cDNA cloning vector with automatic subcloning capability. cDNA libraries constructed in these vectors were used to isolate genes from humans and Arabidopsis thaliana by complementation of yeast and bacterial mutations, respectively.

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox2 plasmids) are cyclized in Cre+ E. coli strains after introduction either by transformation or by mini-Mu transduction. Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox2 plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox2 plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre+ E. coli recBC strains allow the efficient recovery of lox2 plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.

Site-specific insertion of DNA into a pseudorabies virus vector.

A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.