First, the enhanced green fluorescent protein gene egfp and chloramphenicol resistance gene cm r, along with the two lox p loci, were integrated into the genome of a. In this study we adapted the cre lox recombination system to create large deletions in the b. Using cre lox, we systematically investigated the contribution of 14 adjacent genes on the left arm of lp54 to the overall infectivity of b. Crelox recombination is a sophisticated sitespecific recombinase technology that allows dna modification to be targeted to a specific cell type or to be triggered by a specific external stimulus. However, this system requires many timeconsuming steps, including construction of a cre expression plasmid, introduction of the plasmid, and cre. Crelox recombination is a sitespecific recombinase technology, used to carry out deletions. Aug 15, 2019 transgenic mice are produced for several experiments by manipulating genes by overexpressing it, knocking out or addition of a new gene etc. To use cre lox technology, an investigator has to produce a cre lox mouse, typically by breeding a cre mouse to a loxp mouse.
Crelox system and pcrbased genome engineering in bacillus. Jun 21, 2017 the cre loxp recombination system is routinely used for the generation of mouse knockouts. Construction of markerfree transplastomic tobacco using the creloxp sitespecific recombination system. Sep 16, 2019 upon cre recombinasemediated recombination, the loxp sites are recombined resulting in the excision of the 3 tandem sv40 polyadenylation signals. Nov 25, 2015 this video discusses the mechanisms behind the cre loxp recombination, which has been used in many research studies to manipulate genes. The cre gene, which codes for a sitespecific recombinase, was placed under control of the yeast gali promoter. The cre loxp recombination system is widely used as a genetic tool to achieve conditional gene expression and for lineage tracing. The use of cre recombinase to carry out conditional mutagenesis of transgenes and insert dna cassettes into eukaryotic chromosomes is widespread. This is explained by crossing over between the gene pairs during meiosis in the parents. Sitespecific gene recombination is achieved by targeting loxp recognition sites that flank the. Specifically, we use the example of excising a lacz stop. Construction of markerfree transplastomic tobacco using the creloxp site specific recombination system. To minimize genetic instability, a pair of mutant lox sites fig. A cre mouse contains a cre recombinase transgene under the direction of a tissuespecific promoter.
When cremediated recombination in mice does not result in. It is a member of a large family of tyrosine recombinases that includes several other familiar recombinases, such as flp and int. Verma, august 7, 2001 the cre recombinase cre from bacteriophage p1 is an important. Pdf construction of markerfree transplastomic tobacco.
Crelox technology and breeding schemes for research. Cre lox system derived from p1 bacteriaphage can quickly and effectively achieve gene insertion, deletion, replacement, and inversion by means of sitespecific recombination. Cre mediated recombination can be achieved by crossing floxed mice to another mouse line expressing cre recombinase along with a fluorescent marker in a subset of cells cre reporter line. Research article open access development of selectable marker.
In addition, recombination systems such as cre lox have been used extensively, mainly for eukaryotic organisms. Genetic recombination and conditional knockouts practice. Our data suggest that bac ins1cre25 mice are a useful cre driver c57bl6n for pancreatic. We compared the in vivo cre mediated recombination efficiencies of different lox. Longer incubation times will not improve recombination. Construction of markerfree genetically modified maize. Research article open access development of selectable. Functional expression of the crelox sitespecific recombination system in the yeast saccharomyces cerevisiae. Creloxp recombination essentials part 1 bitesize bio. The crelox and flpfrt systems the jackson laboratory. Cre recombinasemediated sitespecific recombination between plant.
At the start of the recombination pathway, the loxp sites are each bound by two recombinase molecules, and synapsis of the sites is mediated by crecre interactions. This video discusses the mechanisms behind the cre loxp recombination, which has been used in many research studies to manipulate genes. Cre lox recombination is a special type of sitespecific recombination developed by dr. Crelox recombination an overview sciencedirect topics. Production of markerfree transgenic rice expressing tissuespecific bt gene. Construction of markerfree transplastomic tobacco using the cre. Cre lox recombination is a sitespecific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the dna of cells. Media in category cre lox recombination the following 9 files are in this category, out of 9 total. Vikavox, a novel efficient and specific creloxplike. Delivery of the cre recombinase by a selfdeleting lentiviral vector. Efficient gene targeting in vivo alexander pfeifer, eugene p. The crelox recombination system is one of the best characterized and most widely.
The first part of this protocol was modified to allow for cre lox recombination. Modified creloxp recombination in aspergillus oryzae by. This is a pdf file of an unedited manuscript that has been accepted for publication. Random integration of lox sites into the es genome was chosen rather than targeted integration because the effect of chromatin structure on cremediated recombination efficiency in vivo is unknown. To gain insight into the ubiquitous recombination efficiency of fgfr1. The use of cre in cellfree systems for construction of recombinant vectors has also been reported, 43. This translocation should produce two new transgenes. Cre recombinasemediated sitespecific recombination between.
Jul 22, 2000 crelox recombination occurred between the two vectors to give the desired result of targeted integration of fulllength yacs. Thus, marker free transgenic citrus plants with targeted traits can be efficiently produced using a cre loxpmediated recombination system combined with ipt positive selection. The procaryotic cre lox sitespecific recombination system of coliphage p1 was shown to function in an efficient manner in a eucaryote, the yeast saccharomyces cerevisiae. Creloxbased system for multiple gene deletions and. A photoactivatable cre loxp recombination system for. Flpfrt recombination system from saccharomyces cerevisiae, where the flp recombinase acts on the frt sites 3. Cre lox recombination involves the targeting of a specific sequence of dna and splicing it with the help of an enzyme called cre recombinase. Detecting and avoiding problems when using the crelox system. Instead, this will likely lead to higher molecular weight recombination products. Construction of adenovirus vectors through crelox recombination. Cloning of a dna fragment from the lefthand terminus of the adenovirus type 2 genome and its use in sitedirected mutagenesis.
The cre protein encoded by the coliphage p1 is a 38kda protein that efficiently promotes both intra and intermolecular synapsis and recombination of dna both in escherichia coli and in vitro. Yet over the past 20 years, cre lox recombination mediated neurogenetics has emerged as one of the most powerful and versatile technology platforms for cellspecific gene knockouts, transgenic. Application of creloxp recombination for lineage tracing. Crelox recombination system for the targeted integration of. Get a printable copy pdf file of the complete article 1. Asymmetric dna bending in the creloxp sitespecific. Typically, only one copy of the cre transgene is required for efficient recombination. Reciprocal crosses were made between t 1 asal lox hpt lox and cre bar plants.
The cre loxp recombination system is a classical tool for targeted genetic engineering. Sitespecific recombination strategies for engineering. It is implemented both in eukaryotic and prokaryotic systems. Ectopic germline recombination activity of the widely used. To test whether vika shows activity on target sites of other known recombinases, we cloned its coding sequence into e. The more complex dna recognition requirement and irreversibility of the.
A cre lox mediated recombination using crossing strategy was adopted to remove the hpt gene from the subsequent generation of selected hybrid events. Increasing the amount of cre recombinase in the reaction can inhibit recombination by forming loxp dependent cre dna aggregates. In addition to the predicted transgene excision event, two alternative pathways of cre. We chose to employ the well characterized bacteriophage crelox. Marker rescue is an important molecular technique that enables sequential gene deletions. Use of the crelox recombination system to develop a nonlethal. Pdf crelox technologies for plant transformation researchgate. C31 system may make it a valuable recombination system for delivering. The illuminacre lox matepaired library preparation protocol presented here is similar to the illumina mate pair library v2 sample preparation guide for 25 kb libraries.
Application of the cre lox p sitespecific recombination. The crelox recombination approach is commonly used to generate. Cre lox recombination is a sitespecific recombinase technology, used to carry out deletions. The linkage is not always complete, meaning that nonparental genotypes are seen in a proportion of the progeny. Pdf the crelox system to assess the development of the. Recombination of dna assortment, leading to the conclusion that those genes are linked on a chromosome. The cre lox recombination approach is commonly used to generate cellspecific gene inactivation or activation. Handbook of pharmacogenomics and stratified medicine, 2014.
In this genetic binary system, cre recombinase catalyzes sitespecific recombination. Cre lox recombinasemediated cassette exchange for reversible sitespecific genomic targeting of the disease vector, aedes aegypti. Delivery of the cre recombinase by a selfdeleting lentiviral. These methods include transient expression of cre albert et al. Detecting and avoiding problems when using the crelox. Cre lox technology and breeding schemes for research emily l. Target gene may be expressed prior to cre recombination if possible, have cre. Use of the crelox recombination system to develop a non. Recently, the cre lox recombination system has become a major technology for modification of gene expression 4, 5. Because systemic inactivation of many genes further cause embryonic lethality, cre lox recombination is commonly used to circumvent this problem. Use of the system has greatly expanded our ability to precisely. Many cre lines now exist for distinct tissue and neural cell types. The classic strategy to achieve gene deletion variants is based on doublecrossover integration of nonreplicating vectors into the genome.
Sitespecific dna recombination in mammalian cells by the. This technology is often used in the generation of knockout and conditional knockout animals. Most investigators breed a new cre recombinaseexpressing cre driver line of mice with a reporter line of mice in which expression of the reporter is activated by deleting a lox stop lox sequence to test for the specificity of the cre induced recombination. The cre or cre recombinase protein called so because it causes recombination catalyzes sitespecific recombination events between two dna recognition sites. To functionally test mitotic recombination in vivo, a mouse line was generated in which mitotic recombination could be induced by two different site specific recombinases, flp and cre.
An alternative to the cre lox system is the flpfrt recombination system. Construction of marker free transplastomic tobacco using the creloxp sitespecific recombination system. Recombination of lox71 and lox66 results in a doublemutant lox72 site that has a strongly reduced binding af. A novel conditional zsgreenexpressing transgenic reporter. Request pdf flpfrt and crelox recombination technology in c. The lox or loxp sites locus of x over p1 are the palindromic sites recognized by cre. In addition to the numerous in vivo and in vitro applications that have been reported since cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the cre. Pdf progress in crelox sitespecific recombination system in. Inducing crelox recombination in mouse cerebral cortex.
Recombination occurs at a specific site, called lox, and does not require any other protein factors. Illumina matepaired dna sequencinglibrary preparation. Functional expression of the cre lox sitespecific recombination system in the yeast saccharomyces cerevisiae. Cre catalyzes recombination between the lox sequences, excising the target gene from the genome. Cre recombinase, isolated from bacteriophage p1, is a 38kda protein, which is able to catalyze conservative reciprocal recombination in mammalian cells 6. Creloxp recombination essentials part 2 bitesize bio. Cre recombinase allows cellspecific manipulation of gene expression.
Jun 01, 2017 the cre or cre recombinase protein called so because it causes recombination catalyzes sitespecific recombination events between two dna recognition sites. These genes should be regulated and would be more useful if expressed in a tissuespecific manner. Construction of marker free transplastomic tobacco using the cre loxp sitespecific recombination system. Cre loxp recombination enables cellular specificity and, in the case of inducible systems, temporal control of genomic deletions. Marker gene elimination was confirmed in the resulting f 1 hybrid progenies by pcr analysis, using hpt, cre. For additional usage notes, please see the cre recombinase product page. Use of the crelox recombination system to investigate the.
This video describes about cre lox recombination system in details. The cre loxp recombination system has been used for marker gene rescue in various organisms, including aspergilli. Furthermore, both the cre and flp proteins promote efficient recombination in vitro without the requirement for any accessory proteins. We describe the structures of synaptic complexes formed between a symmetrized loxp site and two cre mutants that. Cre shown at left is a bacteriophage p1 member of the integrase family, catalyzing sitespecific recombination between two, 34base pair lox dna sequences. Crelox recombination is a special type of sitespecific recombination developed by dr. The crelox system to assess the development of the mouse brain chapter pdf available in methods in molecular biology clifton, n.
A targetronrecombinase system for largescale genome. The crelox recombination system is derived from the bacteriophage p1. In part 1 of this miniseries, i introduced the concept and applications of cre loxp. Genetic recombination and conditional knockouts if youre seeing this message, it means were having trouble loading external resources on our website. We utilized the cre lox recombination system to develop the first knockin murine model for osteogenesis imperfecta oi. Bluelightinduced dimerization of a split cre system enables efficient lightcontrolled dna integration. It allows the dna modification to be targeted to a specific cell type or be triggered by a specific external stimulus.
Use of the system has greatly expanded our ability to precisely interrogate gene function in the mouse, providing both spatial and temporal control of gene expression. Cre catalyzes reciprocal recombination between its lox sites. The cre lox recombination system is derived from the bacteriophage p1. However, there was no cre loxp recombination mediated reporter signal in the brain of both f 1 mice. This study presents the construction of a cre lox based system for multiple gene deletions in lactobacillus plantarum that could be adapted.
Conditional gene expression using the cre lox flex vector. Vergunst and hooykaas, 1998 and use of a cre promoterdisplacement strategy albert et al. The lox sites are similar to the flp recognition target sites of flp in both overall architecture and actual sequence 25. Cre recombinase has low affinity for the double mutant loxlere site, which is generated by cre mediated recombination between loxle and loxre together with a wildtype loxp site. We have noticed that the breeding and genotyping sections of papers utilizing cre lox. The authors propose that the reason for this is twofold. The cre loxp system, derived from p1 bacteriophage, is a sitespecific recombination system extensively used in experimental organisms 1, 2. Cre recombinaseloxp recognition site system from bacteriophage p1, where the cre enzyme recognizes its specific target sites 2. The lox or loxp sites locus of x over p1 are the palindromic sites recognized by cre recombinase. Jamey marth showed that crelox recombination could be used to delete loxpflanked chromosomal dna sequences at high efficiency in selected cell types of. Cre recombinase catalyzes sitespecific recombination between two 34bp loxp sites in a variety of dna substrates. A rapid in vitro method to flip back the doublefloxed. Brian sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice. Marker gene elimination was confirmed in the resulting f 1 hybrid progenies by pcr analysis, using hpt, cre and asal.
Target gene may be expressed prior to cre recombination consider that genetic background may affect phenotype heffner et al. The cre loxp recombination system is routinely used for the generation of mouse knockouts. If youre behind a web filter, please make sure that the domains. C31mediated recombination is unidirectional and irreversible. As with any other technology or research tool, it has limitations and pitfalls that need to be considered while planning experiments or interpreting results.
522 643 770 850 118 781 649 49 615 704 1121 914 1080 291 640 165 1167 527 1008 1517 928 317 130 100 27 906 1459 843 179 883 555 290 156 953 1377