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Cre recombinase

Cre recombinase is a site-specific DNA recombinase derived from the P1 bacteriophage.
It catalyzes the site-specific recombination between two 34-base pair sequences known as loxP sites, allowing for precise genetic manipulations such as gene knockout, insertion, or conditional expression.
Cre recombianase is widely used in research to generate genetically modified organisms and cell lines, enabling the study of gene function and disease pathways.
Its versatility and precision make it an indispensable tool for advanced molecular biology and genetics investigations.

Most cited protocols related to «Cre recombinase»

Sparse Labeling of Cortical Neurons for Imaging

Cited 183 times

Constructs used to produce AAV included pGP-AAV-syn-GCaMP-WPRE and the Cre recombinase-activated construct pGP-AAV-syn-flex-GCaMP-WPRE. Virus was injected slowly (30 nL in 5 minutes) at a depth of 250 μm into the primary visual cortex (two sites, 2.5 and 2.9 mm lateral from the lambda suture). For population imaging and electrophysiology (Fig 2-3), AAV2/1-syn-GCaMP-WPRE virus (titer: ∼10^{11 (link)} -10^{12 (link)} genomes/mL) was injected into the visual cortex of C57BL/6J mice (1.5-2 months old)^{6 (link)}. For dendritic imaging (Fig 4, 5 and 6a-f), sparse labeling was achieved by injecting a mixture of diluted AAV2/1-syn-Cre particles (titer: ∼10^{12 (link)} genomes/mL, diluted 8000-20,000 fold in PBS) and high titer, Cre-dependent GCaMP6s virus (∼8×10^{11 (link)} genomes/mL). This produces strong GCaMP6 expression in a small subset of neurons (∼3-5 cells in a 250 μm × 250 μm × 250 μm volume), defined by Cre expression^{56 (link)}. Both pyramidal (Fig. 4-5) and GABAergic (Fig. 6) neurons were labeled using this approach, but they could be distinguished based on the presence or absence of dendritic spines. Post hoc immunolabeling further identified the imaged cells. For specific labeling of parvalbumin interneurons (Fig. 6g and Supplementary Fig. 12), Cre-dependent GCaMP6s AAV was injected into the visual cortex of PV-IRES-Cre mice^{57 (link)}. Individual somata (Supplementary Fig. 12) and dendritic segments could be recognized (Fig. 6 g, h, total length of imaged dendrite: 2.86 mm), but the high labeling density made it difficult to track individual dendrites over long distances.

Chen T.W., Wardill T.J., Sun Y., Pulver S.R., Renninger S.L., Baohan A., Schreiter E.R., Kerr R.A., Orger M.B., Jayaraman V., Looger L.L., Svoboda K, & Kim D.S. (2013). Ultra-sensitive fluorescent proteins for imaging neuronal activity. Nature, 499(7458), 295-300.

Publication 2013

Cells Cre recombinase Dendrites Dendritic Spines Genome Internal Ribosome Entry Sites Interneurons Mice, Inbred C57BL Neurons Parvalbumins Striate Cortex Sutures TCL1B protein, human Virus Visual Cortex

Generation of CD11c-Cre Transgenic Mice

Cited 113 times

To generate the CD11c-Cre transgene, the 160-kb mouse genomic BAC clone RP24-361C4 (BACPAC Resources) was modified by ET recombination, as previously described (43 (link)). The clone contains the entire Itgax (CD11c) gene but lacks the 5′ end of the adjacent Itgam (CD11b) gene, preventing the overexpression of the latter. The recombination cassette containing the Cre recombinase open reading frame, followed by the bovine growth hormone (BGH) polyA signal and the FRT site-flanked prokaryotic Zeocin resistance cassette (Zeo^R), replaced the coding part of the first CD11c exon, and the Zeo^R cassette was subsequently removed by FLP-mediated recombination. The clone insert was released from the vector backbone using NotI digestion, gel-purified, and microinjected into fertilized oocytes. The founder line containing two copies of the transgene (as determined by quantitative Southern hybridization) was chosen for further analysis. Mice were genotyped by genomic PCR using either generic Cre primers or primers specific for the CD11c-Cre transgene (5′-ACTTGGCAGCTGTCTCCAAG-3′ and 5′-GCGAACATCTTCAGGTTCTG-3′ were specific for the CD11c promoter and Cre, respectively).
The R26-EYFP strain (21 (link)) was provided by F. Costantini (Columbia University, New York, NY). The RBP-J^fl strain (19 (link)) was provided by L. Hennighausen (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD), with permission from T. Honjo (Kyoto University, Kyoto, Japan). The Mx1-Cre strain was previously described (44 (link)). Cre-negative RBP-J^fl/fl littermates of CKO (RBP-J^fl/fl Cre⁺) mice were used as controls; in preliminary experiments, wild-type CD11c-Cre⁺ mice were used as controls and were found indistinguishable from CD11c-Cre⁻ animals. For inducible RBP-J deletion, adult RBP-J^fl/fl Mx1-Cre⁺ or control RBP-J^fl/fl mice were injected with 0.25 mg poly(I):(C) three times, with 2-d intervals, and analyzed 3 wk later. For hematopoietic reconstitution, 3 × 10⁶ total BM cells per mouse were injected i.v. into lethally irradiated C57BL/6 mice congenic for CD45.1. The recipient mice were analyzed 4–5 wk after reconstitution. Mice were maintained in a specific pathogen-free facility and used according to the protocol approved by the Columbia University's Institutional Animal Care and Use Committee.

Caton M.L., Smith-Raska M.R, & Reizis B. (2007). Notch–RBP-J signaling controls the homeostasis of CD8− dendritic cells in the spleen. The Journal of Experimental Medicine, 204(7), 1653-1664.

Publication 2007

Adult Animals Cells Clone Cells Cloning Vectors Cre recombinase Crossbreeding Deletion Mutation Diabetes Mellitus Digestion Digestive System Exons Generic Drugs Genes Genome growth hormone, bovine Hematopoietic System Institutional Animal Care and Use Committees ITGAM protein, human Kidney Diseases Mice, Inbred C57BL Mice, Laboratory Oligonucleotide Primers Ovum Poly A Poly I-C Prokaryotic Cells RBPJ protein, human Recombination, Genetic Specific Pathogen Free Strains Transgenes Vertebral Column Zeocin

CRISPR-Cas9 Genome Editing in C. elegans

Cited 92 times

Plasmids have been deposited in Addgene with the following accession numbers: Cas9-sgRNA plasmid targeting a site near ttTi5605, #47550; Cas9-sgRNA plasmid with no targeting sequence, #47549; Peft-3::Cre::tbb-2 3’UTR construct, #47551. All other plasmids used in this study are available from the authors upon request.
To construct the Cas9-sgRNA expression plasmid shown in Fig. 1c, we first designed a synthetic gene encoding Cas9, with C. elegans coding bias and synthetic C. elegans introns, using the C. elegans Codon Adapter^{40 (link)}. Our Cas9 sequence includes a Nuclear Localization Signal and an HA tag at the C-terminus. The synthetic gene was produced as a series of overlapping 500 bp gBlocks (Integrated DNA Technologies), assembled using Gibson Assembly (New England BioLabs) and inserted into the vector pCFJ601 (Peft-3::Mos1 Transposase::tbb-2 3’UTR)^{17 (link)} in place of the Mos1 transposase. Next, a gBlock containing the U6 promoter and sgRNA sequence was inserted 3’ of the tbb-2 3’UTR. Genomic targets of Cas9 conform to the target sequence GN₁₉NGG, where N is any base. The initial G is a requirement for transcription initiation by the U6 promoter, and the NGG (PAM) motif is required for Cas9 activity (note that the NGG motif must be present in the genomic target but is not included in the sgRNA sequence). To target Cas9 to different genomic sequences, we inserted the desired targeting sequence into the Cas9 + sgRNA construct using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) with forward primer 5’-N₁₉GTTTTAGAGCTAGAAATAGCAAGT-3’, where N₁₉ is replaced by the desired 19 bp targeting sequence, and reverse primer 5’-CAAGACATCTCGCAATAGG-3’. Supplementary Table 5 lists the targeting sequences used in this study.
Targeting vectors for single-copy transgene insertion on chromosome II were constructed in the pCFJ150 vector backbone^{20 (link)} using Gateway cloning. We used site-directed mutagenesis with the Q5 site-directed mutagenesis kit (New England Biolabs) to delete a short region of the 3’ recombination arm comprising the Cas9 target sequence, to prevent the homologous repair templates from being cleaved by Cas9.
Homologous repair templates for GFP insertion and lin-31 mutagenesis were constructed in two steps. First, we PCR amplified a 3–4 kb region centered on the desired modification from N2 genomic DNA and cloned the resulting fragment into the pCR-Blunt vector using the ZeroBlunt TOPO Cloning Kit (Life Technologies). Second, we modified this genomic clone by inserting GFP (for GFP knock-ins) or a 3’ exon containing point mutations (for lin-31 mutagenesis), along with the unc-119(+) rescue gene flanked by LoxP sites. GFP and unc-119(+) fragments were generated by PCR, and LoxP sites were included in the unc-119(+) primers. The mutated lin-31 3’ exons were synthesized as gBlocks. These fragments were integrated into the genomic clones using Gibson assembly, which allows for seamless fusion of DNA fragments without the need to include any extra sequence (e.g. restriction sites). To avoid cleavage of the repair templates by Cas9, we deleted or mutated the Cas9 target site in all repair templates. Complete plasmid sequences of all targeting vectors are available from the authors upon request.
To construct the Peft-3::Cre::tbb-2 3’UTR plasmid used for removal of selectable markers with Cre recombinase, we first amplified the Cre ORF from the plasmid pEM3 (ref. 41 (link)) and cloned it into the Gateway donor vector pDONR221. We then performed a 3-fragment gateway reaction using our Cre donor vector, pCFJ386 (Peft-3; a gift from Christian Frøkjær-Jensen), pCM1.36 (tbb-2 3’UTR)^{42 (link)} and the destination vector pCFJ212 (ref. 17 (link)), which contains an unc-119(+) rescue gene.
Supplementary Table 6 lists all primers used in this study.

Dickinson D.J., Ward J.D., Reiner D.J, & Goldstein B. (2013). Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination. Nature methods, 10(10), 1028-1034.

Publication 2013

3' Untranslated Regions Caenorhabditis elegans Chromosomes Cloning Vectors Codon Cre recombinase Cytokinesis Exons Genes Genome Introns Mos1 transposase Mutagenesis Mutagenesis, Site-Directed Nuclear Localization Signals Oligonucleotide Primers Plasmids Point Mutation Recombination, Genetic Synthetic Genes Tissue Donors Topotecan Transcription Initiation, Genetic Transgenes

Generating Cre-expressing Mouse Lines

Cited 72 times

Protocol full text hidden due to copyright restrictions

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Publication 2011

Animals Animals, Laboratory Antibiotics Bacteria Cholinergic Neurons Clone Cells Codon, Initiator Codon, Terminator Cre recombinase FLP recombinase Genes Genome Institutional Animal Care and Use Committees Mice, Inbred C57BL Mice, Laboratory Neomycin Neurons Recombination, Genetic Ribosomes

Generation and Characterization of Cre-Expressing Transgenic Mice

Cited 51 times

Splicer mice were generated with a transgene from pTet-Cre, which contains the Cre recombinase coding sequence from pBS185 (GIBCO BRL) cloned into the EcoRV site of pTet-Splice (GIBCO BRL) as a Klenow-blunted MluI-XhoI fragment. TIE2Cre transgenes were generated with a TIE2 kinase promoter/enhancer cassette described previously 53. The construct pSPTg.T2FXK (pg54) (a gift from Thomas Sato, Beth Israel Hospital, Boston, MA) contained the TIE2 kinase promoter and enhancer with HindIII and NotI sites between the two. These sites allowed us to directionally clone a HindIII-NotI fragment from pTet-Cre, thus introducing the Cre recombinase coding sequence, intronic donor/acceptor sequences, and polyadenylation signal sequences into pg54. This TIE2Cre transgene was then excised from the vector backbone using SalI.
All transgenic mice were generated on a (C3H × C57BL/6)F₂ background. Screening of tail DNA for Cre recombinase transgene presence was by PCR with the following primers: forward, 5′-CGATGCAACGAGTGATGAGG-3′; and reverse, 5′-CGCATAACCAGTGAAACAGC-3′. Positive founder mouse lines were then crossed with C57BL/6 mice for two generations before interbreeding with VCAM-1 knock-in mice.

Koni P.A., Joshi S.K., Temann U.A., Olson D., Burkly L, & Flavell R.A. (2001). Conditional Vascular Cell Adhesion Molecule 1 Deletion in Mice: Impaired Lymphocyte Migration to Bone Marrow. The Journal of Experimental Medicine, 193(6), 741-754.

Publication 2001

Clone Cells Cloning Vectors Cre recombinase DNA Polymerase I Introns Mice, Inbred C57BL Mice, Laboratory Mice, Transgenic Oligonucleotide Primers Open Reading Frames Phosphotransferases Polyadenylation Signal Peptides Tail Tissue Donors Transgenes Vascular Cell Adhesion Molecule-1 Vertebral Column

Most recents protocols related to «Cre recombinase»

Biosynthesis of Bisbenzylisoquinoline Alkaloids in Engineered Yeast

Not available on PMC !

Example 5

FIG. 16 illustrates (A) a biosynthetic scheme for conversion of L-tyrosine to bisBlAs and (B) yeast strains engineered to biosynthesize bisBlAs, in accordance with embodiments of the invention. In particular, FIG. 16 illustrates (A) a pathway that is used to produce bisBlAs berbamunine and guattegaumerine. FIG. 16 provides the use of the enzymes ARO9, aromatic aminotransferase; ARO10, phenylpyruvate decarboxlase; TyrH, tyrosine hydroxylase; DODC, DOPA decarboxylase; NCS, norcoclaurine synthase; 6OMT, 6-O-methyltransferase; CNMT, coclaurine N-methyltransferase; CYP80A1, cytochrome P450 80A1; CPR, cytochrome P450 NADPH reductase. Of the metabolites provided in FIG. 16, 4-HPA, 4-HPP, and L-tyrosine are naturally synthesized in yeast. Other metabolites that are shown in FIG. 16 are not naturally produced in yeast.

In examples of the invention, a bisBIA-producing yeast strain, that produces bisBlAs such as those generated using the pathway illustrated in (A), is engineered by integration of a single construct into locus YDR514C. Additionally, FIG. 16 provides (B) example yeast strains engineered to synthesize bisBlAs. Ps6OMT, PsCNMT, PsCPR, and BsCYP80A1 were integrated into the yeast genome at a single locus (YDR514C). Each enzyme was expressed from a constitutive promoter. The arrangement and orientation of gene expression cassettes is indicated by arrows in the schematic. These strains convert (R)- and (S)-norcoclaurine to coclaurine and then to N-methylcoclaurine. In one example, the strains may then conjugate one molecule of (R)—N-methylcoclaurine and one molecule of (S)—N-methylcoclaurine to form berbamunine. In another example, the strains may conjugate two molecules of (R)—N-methylcoclaurine to form guattegaumerine. In another example, the strains may conjugate one molecule of (R)—N-methylcoclaurine and one molecule of (S)-coclaurine to form 2′-norberbamunine. In another embodiment, the strain may be engineered to supply the precursors (R)- and (S)-norcoclaurine from L-tyrosine, as provided in FIG. 5.

The construct includes expression cassettes for P. somniferum enzymes 6OMT and CNMT expressed as their native plant nucleotide sequences. A third enzyme from P. somniferum, CPR, is codon optimized for expression in yeast. The PsCPR supports the activity of a fourth enzyme, Berberis stolonifera CYP80A1, also codon optimized for expression in yeast. The expression cassettes each include unique yeast constitutive promoters and terminators. Finally, the integration construct includes a LEU2 selection marker flanked by loxP sites for excision by Cre recombinase.

A yeast strain expressing Ps6OMT, PsCNMT, BsCYP80A1, and PsCPR is cultured in selective medium for 16 hours at 30° C. with shaking. Cells are harvested by centrifugation and resuspended in 400 μL breaking buffer (100 mM Tris-HCl, pH 7.0, 10% glycerol, 14 mM 2-mercaptoethanol, protease inhibitor cocktail). Cells are physically disrupted by the addition of glass beads and vortexing. The liquid is removed and the following substrates and cofactors are added to start the reaction: 1 mM (R,S)-norcoclaurine, 10 mM S-adenosyl methionine, 25 mM NADPH. The crude cell lysate is incubated at 30° C. for 4 hours and then quenched by the 1:1 addition of ethanol acidified with 0.1% acetic acid. The reaction is centrifuged and the supernatant analyzed by liquid chromatography mass spectrometry (LC-MS) to detect bisBlA products berbamunine, guattegaumerine, and 2′-norberbamunine by their retention and mass/charge.

US11859225B2. Methods of producing epimerases and benzylisoquinoline alkaloids (2024-01-02). The Board of Trustees of the Leland Stanford Junior University [US]. Inventors: Christina D. Smolke [US], Stephanie Galanie [US], Isis Trenchard [US], Catherine Thodey [US], Yanran Li [US].

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Patent 2024

2-Mercaptoethanol 3-phenylpyruvate Acetic Acid Allopurinol Anabolism Barberry Base Sequence berbamunine Buffers Cells Centrifugation coclaurine Codon Cre recombinase Culture Media Cytochrome P450 Dopa Decarboxylase enzyme activity Enzymes Ethanol Gene Expression Genome Glycerin guatteguamerine higenamine Liquid Chromatography Mass Spectrometry Methyltransferase NADP NADPH-Ferrihemoprotein Reductase norcoclaurine synthase Plants Protease Inhibitors Retention (Psychology) S-adenosyl-L-methionine coclaurine N-methyltransferase S-Adenosylmethionine Saccharomyces cerevisiae Strains Transaminases Tromethamine Tyrosine Tyrosine 3-Monooxygenase

Generation and Characterization of Orai Knockout Mice

All animal experiments were carried out in compliance with the Institutional Animal Care and use Committee (IACUC) guidelines of the Pennsylvania State University College of Medicine. All mice were housed under specific pathogen-free conditions and experiments were performed in accordance with protocols approved by the IACUC of the Pennsylvania State University College of Medicine. Mb1-Cre mice (Hobeika et al., 2006 (link)) were obtained from The Jackson Laboratory (strain#: 020505; https://www.jax.org/strain/020505). Orai1^fl/fl mice (Ahuja et al., 2017 (link)) were obtained from Dr. Paul Worley (Johns Hopkins University). Orai3^fl/fl mice were generated by our laboratory through the MMRC at the University of California Davis. A trapping cassette was generated including ‘SA-βgeo-pA’ (splice acceptor-beta-geo-polyA) flanked by Flp-recombinase target ‘FRT’ sites, followed by a critical Orai3 coding exon flanked by Cre-recombinase target ‘loxP’ sites. This cassette was inserted within an intron upstream of the Orai3 critical exon, where it tags the Orai3 gene with the lacZ reporter. This creates a constitutive null Orai3 mutation in the target Orai3 gene through efficient splicing to the reporter cassette resulting in the truncation of the endogenous transcript. Mice carrying this allele were bred with FLP deleter C57BL/6 N mice to generate the Orai3^fl/fl mouse. All experiments were performed with 8–12 week-old age and sex-matched mice.

Emrich S.M., Yoast R.E., Zhang X., Fike A.J., Wang Y.H., Bricker K.N., Tao A.Y., Xin P., Walter V., Johnson M.T., Pathak T., Straub A.C., Feske S., Rahman Z.S, & Trebak M. (2023). Orai3 and Orai1 mediate CRAC channel function and metabolic reprogramming in B cells. eLife, 12, e84708.

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Publication 2023

Alleles Cre recombinase Exons FLP recombinase Genes, vif Institutional Animal Care and Use Committees Introns LacZ Genes Mice, Inbred C57BL Mice, Laboratory Null Mutation Pharmaceutical Preparations Poly A Specific Pathogen Free Strains

Conditional Plp1 Knockout in Mouse Forebrain

To delete the Plp1 gene in the forebrain we interbred Plp^flox mice in which exon 3 of the Plp1 gene is flanked by loxP sites (Lüders et al., 2019 (link); Lüders et al., 2017 (link); Wang et al., 2017 (link)) with mice expressing Cre recombinase under control of the Emx1 gene promoter (Gorski et al., 2002 (link)) on C57Bl/6N background. Genotyping was as reported previously (Gorski et al., 2002 (link); Lüders et al., 2017 (link)). Experimental male Plp^flox/Y*Emx^IREScre and female Plp^flox/flox*Emx^IREScre mice are termed cKO whereas male Plp^flox/Y and female Plp^flox/flox mice served as controls (Ctrl). Mice were bred and kept in the animal facility of the Max Planck Institute of Experimental Medicine with a 12 hr light/dark cycle and two to five mice per cage. All experiments were performed in accordance with the German Animal Protection Law (TierSchG) and approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES); License numbers were 33.19-42502-04-15/1833 and 33.19-42502-04-18/2803.

Arinrad S., Depp C., Siems S.B., Sasmita A.O., Eichel M.A., Ronnenberg A., Hammerschmidt K., Lüders K.A., Werner H.B., Ehrenreich H, & Nave K.A. (2023). Isolated catatonia-like executive dysfunction in mice with forebrain-specific loss of myelin integrity. eLife, 12, e70792.

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Publication 2023

Animals Cre recombinase Exons Females Gene Expression Regulation Genes Males Mice, Laboratory Prosencephalon

Generation of AvCreERT2:Adam23 Mouse Line

To generate the AvCreERT2:Adam23^LoxP/LoxP mouse line, AvCreERT2 mice were bred with Adam23^LoxP/LoxP mice (Lau et al., 2011 (link)). Tamoxifen activation of Cre-recombinase expressed in Advillin-positive cells resulted in deletion of exon 1 of Adam23. Tamoxifen was administered to 6-wk-old mice by gavage at 1.2 mg per 10 g of body weight, for five consecutive days. This was followed by a 1-wk break, after which administration was repeated for another 5 d. Animals were culled and tissue was collected at appropriate time points after Tamoxifen administration.

Kozar-Gillan N., Velichkova A., Kanatouris G., Eshed-Eisenbach Y., Steel G., Jaegle M., Aunin E., Peles E., Torsney C, & Meijer D.N. (2023). LGI3/2–ADAM23 interactions cluster Kv1 channels in myelinated axons to regulate refractory period. The Journal of Cell Biology, 222(4), e202211031.

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Publication 2023

Animals Body Weight Cells Cre recombinase Deletion Mutation Exons Mus Tamoxifen Tissues Tube Feeding

Generation and Characterization of Genetically Modified Mouse Lines

The Adam23^Δ1/Δ1, Lgi2^Δ1/Δ1, Lgi3^Δ1/Δ1, Lgi2^Δ1/Δ1:3^Δ1/Δ1 genotypes were generated in the Meijer lab using a standardized gene recombination approach, as previously described (Kegel et al., 2014 (link); Jaegle et al., 2003 (link)). The method is summarized here using Lgi2^Δ1/Δ1 as an example (see Fig. S1). A LoxP site was inserted upstream of the first exon whilst an Frt-flanked neo cassette with a 3′ LoxP site was inserted downstream, resulting in the Lgi2^LoxP allele. The Lgi2^Δ1 null allele was then generated through crossing Lgi2^LoxP/+ mice with mice carrying germline Cre recombinase leading to deletion of the first exon and its promoter. The Lgi2^Δ1/+ mice were eventually intercrossed, generating Lgi2^Δ1/Δ1 offspring. Mice of the Lgi2^Δ1/Δ1 and Lgi3^Δ1/Δ1 genotypes developed normally, and their lifespans and fertility were not affected by the mutation. Adam23^Δ1/Δ1 and Lgi2^Δ1/Δ1:3^Δ1/Δ1 mice expressed severe phenotypes, characterized by poor postnatal growth, body tremors seen from second post-natal week, and early lethality (around P12 in the Adam23^Δ1/Δ1 mice and P16 in the Lgi2^Δ1/Δ1:3^Δ1/Δ1).

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Publication 2023

Alleles Cre recombinase Deletion Mutation Exons Fertility Genotype Germ Line Human Body Mice, Laboratory Mutation Phenotype postnatal growth Recombination, Genetic Tremor Vision

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Cre recombinase by New England Biolabs

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Cre recombinase is a site-specific DNA recombinase enzyme that catalyzes the recombination of DNA between specific recognition sequences called loxP sites. It is a useful tool in molecular biology for genetic engineering applications.

What are the key features and benefits of using Cre recombinase in research?

Cre recombinase is a powerful genetic engineering tool that offers several key advantages for researchers. Its ability to catalyze site-specific recombination between loxP sites allows for precise genetic manipulations, such as gene knockouts, insertions, and conditional expression. This enables researchers to study gene function and disease pathways with remarkable precision. The versatility and accuracy of Cre recombinase make it an indespensible tool for advanced molecular biology and genetics investigations.

What are some common challenges or limitations in using Cre recombinase?

While Cre recombinase is a highly versatile tool, there can be some challenges in its implementation. Researchers must carefully design their experiments to ensure the appropriate timing and location of Cre expression to achieve the desired genetic modifications. Off-target effects and leaky expression can be potential issues that require thorough validation. Additionally, the availability of well-characterized loxP-flanked (floxed) target genes and Cre driver lines can sometimes limit the specific applications of Cre recombinase.

Are there different variations or types of Cre recombinase available, and how do they differ?

Yes, there are several variations and types of Cre recombinase that have been developed to meet different research needs. For example, tamoxifen-inducible Cre (CreERT2) allows for temporal control of Cre activity, while tissue-specific Cre drivers can restrict Cre expression to particular cell types or developmental stages. Additionally, mutant Cre variants with increased efficiency or altered recombination specificity have been engineered to further expand the toolkit available to researchers.

What are some of the key applications of Cre recombinase in research?

Cre recombinase has a wide range of applications in research, including: 1. Conditional gene knockouts: Allowing researchers to study the effects of gene deletion in a spatially and temporally controlled manner. 2. Lineage tracing: Enabling the tracking of cell lineages and fate mapping by coupling Cre expression with reporter genes. 3. Inducible gene expression: Facilitating the temporal control of gene expression for studying developmental processes or disease pathways. 4. Genetic mosaic analysis: Generating mosaaic tissues or organisms to investigate cell-cell interactions and the effects of genetic manipulations.

How can PubCompare.ai assist researchers in optimizing their use of Cre recombinase?

PubCompare.ai can be an invaluable tool for researchers working with Cre recombinase. The platform's AI-driven protocol comparisons allow you to efficiently screen the available literature, pre-prints, and patents to identify the most effective Cre recombinase protocols for your specific research goals. By leveraging PubCompare.ai's intelligent analysis, you can pinpoint key differences in protocol effectiveness, enabhling you to choose the best option to ensure reproducibility and accuracy in your Cre recombinase experiments. This can help streamline your research process and maximize the impact of your Cre recombinase-based investigations.

More about "Cre recombinase"

Cre recombinase is a versatile genetic engineering tool derived from the P1 bacteriophage.
It catalyzes site-specific recombination between 34-base pair loxP sequences, enabling precise manipulations such as gene knockout, insertion, or conditional expression.
This powerful recombinase is widely used in research to generate genetically modified organisms and cell lines, facilitating the study of gene function and disease pathways.
Cre's precision and flexibility make it an indispensable resource for advanced molecular biology and genetics investigations.
Researchers often leverage Cre in conjunction with other techniques like Tamoxifen-inducible systems, C57BL/6J mouse models, and cell culture methods involving Lipofectamine 2000 and 4-hydroxytamoxifen in FBS-supplemented media.
By harnessing the capabilities of Cre recombinase, scientists can unravel complex biological processes, model human diseases, and develop innovative therapies.
This powerful tool continues to drive breakthroughs in our understanding of the genome and its role in health and disease.
Whether you're working with Cre in vivo or in vitro, exploring its applications can open new doors to groundbreaking discoveries.