Here, we combined lentiviral gene transfer and the development of a tightly drugcontrolled flp recombinase for the construction of allinone inducible gene expression systems. Applications constitutive expression,mammalian expression,protein biology,protein expression,targeted integration. Highefficiency flp and wc31 sitespecific recombination in. Because control by a single recombinase defines relatively broad classes of cells, we also have generated a flp creresponsive allele that restricts hm3dq to the narrow intersection of two broader recombinase expression domains. Noninvasive optical activation of flp recombinase for. Expression of flp recombinase will excise the cassette as a circular dna molecule. A conditional ready floxed allele can be created by flp recombinase expression in mice carrying this allele. Flpmediated conditional loss of an essential gene to.
Flpin expression vectors are available with the cmv or ef1. The flpin system involves introduction of a flp recombination target frt site into the genome of the mammalian cell line of choice. Generation of isogenic expression cell lines using flpin. Jan 22, 2019 the technique provides expandable utilities for transgene expression systems upon flp recombinase activity in vivo, by designing a viral vector for minimal leaky expression influenced by viral.
Flp recombinasemediated cassette exchange of the frtflanked dv rmce sequences results in the functional reconstitution of the rfp reporter gene along with the insertion of the goi middle panel. Improved properties of flp recombinase evolved by cycling mutagenesis. Flprecombinase expression vector designed for use with. It is analogous to crelox recombination but involves the recombination of sequences between short flippase recognition target frt sites by the recombinase flippase flp derived from the 2 plasmid of bakers yeast. Construction of permanently inducible mirnabased expression. Fiveminute topo cloning the pcdna5frtv5his and psectagfrtv5his topo ta expression kits offer fiveminute cloning of taqamplified pcr products directly into a flpin expression vector.
An expression vector containing your gene of interest is then integrated into the genome via flp recombinasemediated dna recombination at the frt site ogorman et al. Viral t2a peptide is cleaved just after translation, and egfp and flp recombinase localize independently. It is based on the gbtr15 vector figure 1a 27, 28, with augless mrfp mrfp replaced by aug. Plasmid files snapgene software for everyday molecular. The average frequency of flp recombinase mediated sitespecific excision in the two target strains genome was approximately 3. In some embodiments, compositions, methods, kits and systems are provided that relate to a split recombinase system, whereby protein complementation or rejoinder of splitrecombinases is mediated by chemicalinduced dimerization domains cidds or lightinduced dimerization.
The technology described herein relates to controlled chemically or lightinduced rejoinder of splitrecombinases. Aug 01, 2017 sitespecific recombinases are potent tools to regulate gene expression. It is stably integrated at the introduced flp recombinase target frt site in the host genome. Analogous template modules for nterminal tagging can be devised. We also verified delivery of paflp via an aav vector in wt mice by coinfecting paflp with fdioyfp, an exogenous flp reporter, demonstrating a 30. To enable an easy and versatile vector construction strategy, resulting in bacillus vectors carrying flp and frtfgoifrtf3, a synthetic dna segment was designed, by which bspq1 sites were built into a small vector segment placed between frtf and frtf3, so that the recognition sites themselves would be cut away by digestion of the vector. Flpin trex 293 cell line from thermo fisher scientific. Previous techniques based on auxotrophy and antibiotic selection methods were useful for the identification of genes that were expressed at high. C31, are powerful tools for analyzing gene function in vertebrates. Efficient chromosomal gene modification with crisprcas9. Us patent application for flpmediated genomic integration in. The pef5frtv5dest and pef5frtv5dtopo vectors contain the human ef1e promoter for driving expression of the gene of interest. Flp recombinase regulated lacz expression at the rosa26 locus.
Dissociating orexindependent and independent functions. More complex boolean logic gates have been described, utilizing both cre and flpdependent expression cassettes that allow creon, flpon, creoff, flpoff, and all combinations of dualrecombinase logic. Apr 01, 2002 one was the flp expression vector p4bflp r1 replication origin, kanamycin resistance, and lowest copy number among the three from which flp could be rapidly and reversibly turned on from the arabinoseinducible promoter p bad. Vector for constitutive expression of a thermolabile flp recombinase flp f70l in mammalian cells, for use with the flp in system. The topo cloning box contains all of the reagents necessary to clone a taqamplified pcr product topo ta or a pcr product generated with a proofreading enzyme directional topo, including 200ng of topoisomerase iactivated vector, sterile water, dntps, 10x pcr buffer, salt solution, control template and primers, primers for sequencing, and an expression control plasmid. Considerations for the use of cre recombinase for conditional. Flp recombination target frt site for flp recombinasemediated integration of the vector into. One was the flp expression vector p4bflp r1 replication origin, kanamycin resistance, and lowest copy number among the three from which flp could be rapidly and reversibly turned on from the arabinoseinducible promoter p bad.
For specific information on the expression vector and the corresponding positive control vector containing the cat gene, refer to the pcdna5frt vector manual. The critical exons isare thus flanked by loxp sites. Thus, expression is dependent on both the promoter driving the coding region and the promoter driving expression of flp. The constructs shown in figure 6a were transfected together with the flp recombinase expression vector into k562 cells a cr2 nonexpressing cell line into which had previously been integrated a single frt sequence figure 1, lane 1. The flp recombinaseexpressing helper vector psla3flp figure 1d was constructed as described below. It is analogous to crelox recombination but involves the recombination of sequences between short flippase recognition target frt. The other two, p33rdw2 and pbu11, harbored the frtw2rfp frtw2 and mfrt11 lacz. The other four flpin expression vectors offer two timesaving options for cloning into a flpin expression vector. The entire u6 promoter u6 prom mirna cassette is flanked by frt sites, which can be utilized to shut down the system in the presence of flp recombinase. The donor vector dv rmce includes a nonfunctional rfp reporter and the goi expression unit upper panel. Flp recombinase expression plasmids for selection cassette removal the following recombinase expression plasmids are very useful to excise frt or loxpflanked dna stretches such as selection markers during a modification task.
An flp indicator mouse expressing alkaline phosphatase from the rosa26 locus. Alex nielsens lab contains the inserts flp recombinase, rhas, and rhar and is published in sci rep. An expression vector containing your gene of interest is then integrated into the genome via flp recombinasemediated dna recombination at the frt site. Furthermore, we engineer a flpdependent cre driver as a module without leaky cre expression in viral vector system, finally showing. Construction of permanently inducible mirnabased expression vectors using sitespecific recombinases. Allinone inducible lentiviral vector systems based on drug. Expression of flp recombinase provides the flpstop approach both spatial and temporal control. If cre expression occurs without flp expression, a reporter knockout mouse will be created. We also engineered a builtin mechanism to destroy the vector with flp recombinase, if desired.
Integration sitespecific transcriptional reporter gene. Generation of flp in expression cell lines requires cotransfection of the flp in cell line with a flp in expression vector containing your gene of interest e. In the first component the first vector, a wildtype version of the gene is placed between target sequences recognized by flp recombinase from the yeast 2. Flpin 293 trex cell lines is designed for rapid generation of stable cell lines that ensure homogenous expression of your protein of interest from a flpin expression vector. Flp recombinase mediated cassette exchange of the frtflanked dv rmce sequences results. Additional flpin expression vectors are available from invitrogen.
Vector for constitutive expression of a thermolabile flp recombinase flpf70l in mammalian cells, for use with the flpin system. They do not have an expression vector for cre recombinase on a blasticidine or zeocin or hygro backbone i am not interested by puromycine. May 16, 2018 the constructs shown in figure 6a were transfected together with the flp recombinase expression vector into k562 cells a cr2 nonexpressing cell line into which had previously been integrated a single frt sequence figure 1, lane 1. Efficient disruption of zebrafish genes using a gal4. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. Recombinasedependent mouse lines for chemogenetic activation. Compare pog44 flp recombinase expression vector from thermo fisher scientific on. The gal4vp16 component of our vector provides two important advantages over other gbt vectors. The other four flpin expression vectors offer two timesaving options for cloning into a. Our gbtb1 for g ene b reaking t ransposon b ipartite 1 vector is composed of several components that in concert ensure efficient mutagenesis, evaluation of the trapped genes expression profile and enable manipulation of resulting mutant alleles. For example, as we demonstrate with ap flpstop, use of the heatshock promoter to express flp restricts inversion of the flpstop construct to a brief time window, a manipulation that is well suited to developmental studies figure 2 and figure 2. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal mirna sequences using our system. These systems use a sitespecific recombinase integrase in gateway and cre recombinase in creator and echo to allow reliable transfer of a fragment from one vector to another without using restriction. We generated a line of hek293 cells stably expressing a cmvgal the grim.
Es cell transfection assay 20 mg of each linearized ssr expression construct was coelectroporated in a 10. The average frequency of flp recombinasemediated sitespecific excision in the two target strains genome was approximately 3. Before marker excision, expression of the targeted genes will thus be constitutive ordepending on the promoterconditional. Product quantity composition pcdna5frt expression vector 20 ug 40. Despite a number of different transgenes that can mediate dna deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies.
Finally, to further simplify the construction process, we developed a software package that automates. Sitespecific recombinases are potent tools to regulate gene expression. The cassette is composed of an frt site followed by lacz sequence and a loxp site. The plasmids are easily transformed into cells resulting in an activation of the recombinases in vivo. These cells contain a single stably integrated frt site at a transcriptionally active genomic locus. Flp recombinase is provided as needed through the regulated expression of its gene on a plasmid. Feb 17, 2017 expression of flp recombinase provides the flpstop approach both spatial and temporal control. Both recombinases work efficiently in the popular model organism caenorhabditis elegans, but their use in this nematode is still only sporadic. The technique provides expandable utilities for transgene expression systems upon flp recombinase activity in vivo, by designing a viral vector for minimal leaky expression influenced by viral. Integration sitespecific transcriptional reporter gene analysis using. Chromosomal targeting in bacteria using flp recombinase.
In genetics, flpfrt recombination is a sitedirected recombination technology, increasingly used to manipulate an organisms dna under controlled conditions in vivo. Tight control of flp activity was achieved through nterminal fusion with a fkbp12derived conditional destruction domain and a cterminal estrogen receptor b. Flp recombinase expression plasmids for selection cassette. Click on underlined text or outlined graphics to explore. Noninvasive optical activation of flp recombinase for genetic. Oct 23, 2017 to enable an easy and versatile vector construction strategy, resulting in bacillus vectors carrying flp and frtfgoifrtf3, a synthetic dna segment was designed, by which bspq1 sites were built into a small vector segment placed between frtf and frtf3, so that the recognition sites themselves would be cut away by digestion of the vector.
This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and. Flprecombinase expression vector designed for use with the flpin system. Similarly, the wc31o and wc31 coding sequences were blunt cloned into the prosa261 vector to generate mouse strains that broadly express this ssr. The flpfrt system has been used in drosophila and several other organisms to induce somatic recombination, generate chromosomal rearrangements, and as a basis for gene targeting. To achieve orexin neuronspecific expression of flp recombinase, we inserted egfp2aflp just behind the translation initiation site of the hypocretin gene inframe. Because control by a single recombinase defines relatively broad classes of cells, we also have generated a flpcreresponsive allele that restricts hm3dq to the narrow intersection of two broader recombinase expression domains. Niels geijsens lab contains the insert cre recombinase and is published in cell.
This construct contains an egfp nterminal tag to ctcf under control of a cmv promoter, introduced to expression vector pcdna5frt as part of thermo fishers flpin system. It can be readily removed by injection of flp mrna for use of our gene trap alleles with other tissuespecific gfpmarked lines. Noninvasive lightsensitive recombinase for deep brain. Flpstop, a tool for conditional gene control in drosophila. Flp recombinasemediated cassette exchange of the frtflanked dv rmce sequences results. Highefficiency flp and wc31 sitespecific recombination. Exogenous dna is introduced on a cloning vector that contains an frt, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. Dec 19, 2017 the donor vector dv rmce includes a nonfunctional rfp reporter and the goi expression unit upper panel. While the availability of multiple highefficiency ssrs would facilitate a wide array of genomic engineering possibilities, efficient recombination in mammalian cells has only been observed with cre recombinase. Using recombinase based in vivo expression technology. This rearrangement will place the promoter adjacent to the downstream coding regions, converting the transgene to the on state. Dna sitespecific recombinases ssrs such as cre, flpe, and.
Hygromycinresistant populations that arose following the transfections were analyzed by flow cytometry. This first loxp site is followed by a neomycin resistance gene under the control of the human betaactin promoter, sv40 polya, a second frt site and a second loxp site. In some embodiments, compositions, methods, kits and systems are provided that relate to a splitrecombinase system, whereby protein complementation or rejoinder of splitrecombinases is mediated by chemicalinduced dimerization domains cidds or lightinduced dimerization. A targeted fusion protein is designed to increase effective expression level, enabling dosedependent induction of cellular activity in diverse cell types. Price pricing info supplier page view company product page. Cmv promoter for high level expression in mammalian cells. Subsequent cre expression results in a knockout mouse. In particular, the cre cyclization recombination and flp flipase enzymes are widely used to either activate or inactivate genes in a precise spatiotemporal manner. Allinone inducible lentiviral vector systems based on. Advantages of the flp in system use of the flp in system to generate stable expression cell lines provides a number of advantages as described below. The attl1 site can be removed by cre recombinase to produce the constitutively active grim. Invitrogen pog44 flprecombinase expression vector 20g.
Flprecombinase expression vector designed for use with the flpin. Peter freddolinos lab contains the insert flp recombinase and is published in cell syst. Efficient chromosomal gene modification with crisprcas9 and. Promoterdriven mirna expression vectors have emerged as important tools. A schematic representations of the hypocretin gene, targeting vector, and targeted gene.727 1549 477 1578 769 134 1236 891 1101 1089 267 1198 1235 82 1007 799 1047 1310 36 783 614 532 1459 425 1559 641 844 848 962 931 207 547 1245 51 426 991 785