Gateway recombination cloning technology thermo fisher. Inducible cre transgenic mouse strain for skeletal muscle. With the creloxp sitespecific recombination system used in most of our hr targeting vectors, you can get rid of extra sequences, such as selection cassettes, by transiently expressing the cre recombinase. While crelox recombination is a very useful tool, cre expression can be toxic in. Conditional gene deletion using crelox recombination is frequently used in mouse genetics. Over the last few years, development of the creloxp system, which is concerned with recombination mechanisms in the bacteriophage p1, has provided powerful technologies for the. Hard drive cloning software why you need it acronis.
Improved reporter strain for monitoring cre recombinase. We used biorads image lab software to get semiquantitative analysis of. Analysis of conditional gene deletion using probe based. Both organisms have specific recombination sites called. Helper virusfree preparation of aav particles to deliver cre.
Mouse reporter strain for noninvasive bioluminescent. The ires was pcr amplified from pires clontech, mountain view, ca, usa and cloned into pentr3c. The appearance of recombinase activity would then be the consequence of the stepwise assembly, on the loxp site, of a native. In fact, persistence of the episomal crerecombinase product was documented in plants s rivastava and o w 2003 and was also noted to be retained after viral cre infection in mice d origo et. A rapid in vitro method to flip back the doublefloxed. Conditional alleles containing loxp recombination sites, in conjunction with cre recombinase delivered by a variety of means, allows for spatial and temporal control of gene. Increasing the amount of cre recombinase in the reaction can inhibit recombination by forming loxp dependent credna aggregates. Homologous recombination into the eosinophil peroxidase. Home dna modifying enzymes and cloning technologies products cre recombinase. P1 bacteriophage genome were generated and cloned into lambda vectors. Cloning methods recombination cloning systems gateway.
Recv recombinase system for in vivo targeted optogenomic. Selfcleaving 2apeptidelinked reprogramming factors. Cre recombinase gesicles give you this control and much more. A guide to creating and testing new intrsect constructs. Viral vectors remain the most efficient and popular in deriving induced pluripotent stem cells ipscs. Cre recombinase gesicles are cellderived nanovesicles used to deliver active cre recombinase protein directly into target cells. A chimeric cre recombinase with regulated directionality. A dio vector will have the gene of interest cloned in the inverted orientation eg. The enzyme uses a topoisomerase i like mechanism to carry out site specific recombination events. We have used a new genetic strategy based on the cre loxp recombination system to generate large chromosomal rearrangements in lactococcus lactis. Homologous recombination in embryonic stem cells was used to generate gene knock. However, they have different requirements and are used for different types of. Recombinational cloning uses a sitespecific recombinase to allow reliable. Codriver employs a recombinase cascade of dre and drerespondent cre, which processes loxpflanked alleles only when both recombinases are expressed in a predetermined temporal sequence.
Genotyping of transgenic mice was performed by pcr analysis primer sequences, table 1. Methods vector design and construction plasmadna software university of helsinki was utilized for the in silico cloning. Cre recombinase is a sitespecific recombinase isolated from the p1 bacteriophage that catalyzes recombination between two 34bp loxp sites. Robust inducible cre recombinase activity in the human. To generate transgenic mice expressing the cre recombinase in germ cells, presumably in the early stages of meiosis, we inserted the cre cdna within. For translation, it is important to silence or remove the reprogramming factors after induction of.
Modulation of cre recombinase in the in vivo cloning of. Using an efficient gene targeting approach, we developed a novel mouse line that expresses the tetracyclinecontrolled transactivator tta from the constitutively active eef1a1 locus in a cre. When cremediated recombination in mice does not result in. Longer incubation times will not improve recombination. It allows insertion of a transgene to be targeted to a specific. Control over your cre recombinase experiments takara bio. Cre recombinase is a type i topoisomerase from bacteriophage p1 that.
The entry clone is how and where you start your experiment, as it contains your gene of interest or dna fragment flanked. Cre recombinase is a widely used tool in the field of molecular biology. A rapid in vitro method to flip back the doublefloxed inverted open. Use of cre recombinase as a universal restriction enzyme.
Effective use of conditional cre recombinase loxp gene modification requires creexpressing mouse strains with defined patterns of expression. Efficient tissuetype specific expression of target genes. A robust strategy for negative selection of creloxp. The gateway cloning technology is based on the sitespecific recombination system used by phage l to integrate its dna in the e. Crelox recombination is a sitespecific recombinase technology widely used to carry out deletions, insertions, or translocations of genes. Crelox recombination an overview applied biological materials. Connie cepkos lab contains the insert cre and is published in proc natl acad sci u s a. Niels geijsens lab contains the insert cre recombinase and is published in cell. Cre recombinase effectively inverted orf in flex plasmid in vitro. Cre recombinase produced by bacteriophage p1 catalyzes sitespecific recombination of dna between loxp recognition sites in both prokaryotic and eukaryotic cells and has been widely used for.
These systems use a sitespecific recombinase integrase in. To expand credependent tools, the cepko group also developed a split component system that reconstitutes cre recombinase in the presence of gfp tang et al. Cloning vector has the cmv promoter driving expression of cre recombinase. Crelox is a powerful tool for genetic manipulation which allows for excellent. Efficient delivery of crerecombinase to neurons in vivo. Diskcloning software is the perfect way to protect and manage your precious data. Cetin conceptualized the lightinducible recombinase system. Both crispr and creloxp are tools for directing modification to a specific dna sequence within the organism. Learn more about how this product is being used in the product citation tool. Sitespecific recombinational cloning using gateway and in. Recombinational cloning became popular with the introduction of three cloning systems. Expression of cre recombinase during transient phage. We have developed a novel way to use the creloxp system for in vitro manipulation of dna and a technique to clone dna into circular episomes.1537 980 469 129 1022 47 1138 1402 1097 142 1387 1449 1000 1652 1645 1592 1148 1443 1330 1309 631 169 261 198 75 874 1033 183 152 1354 1241 1612 1381 864 1380 837 780 848 821 308 304 664 1453 280 1013