Pcag cre gfp

Manufactured by Addgene

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PCAG-Cre:GFP is a plasmid that contains the Cre recombinase gene fused to the green fluorescent protein (GFP) gene. The Cre recombinase enzyme catalyzes site-specific DNA recombination, and the GFP reporter allows for the detection of Cre expression.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions) Puromycin, by Merck Group (3 mentions) Spcas9 protein, by Aldevron (2 mentions) Accutase, by Merck Group (2 mentions) Puromycin, by Addgene (2 mentions) Pvsv g, by Takara Bio (1 mentions) Ganciclovir, by InvivoGen (1 mentions) Mouse es cell nucleofector kit, by Lonza (1 mentions) Nucleofector 2, by Lonza (1 mentions) Px458 plasmid, by Addgene (1 mentions) Gfp rab7 wt, by Addgene (1 mentions) Pmrfp lc3, by Addgene (1 mentions) Gfp rab7t22n, by Addgene (1 mentions) Pgex 4t 3 mr7bd, by Addgene (1 mentions) Aav9 ef1α dio mcherry, by BrainVTA (1 mentions) Gene pulser 2, by Bio-Rad (1 mentions)

15 protocols using pcag cre gfp

Generation of CRISPR Knockout Cell Lines

Cited 3 times

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The pLentiCRISPRv2-LoxP plasmid was generated by inserting LoxP sites (5′-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3′) flankingthe elongation factor-1 α short promoter of plentiCRISPRv2 (Addgene 52961; Shalem et al., 2014 (link)). gRNA sequences targeting lyspersin (5′-GCCGCAGCTTGACGTCTTCG-3′) and LAMTOR1 (5′-GAGGAGGGACGGCGTCCGTG-3′) were chosen using an online prediction tool (CRISPR Design; Zhang laboratory, MIT; Hsu et al., 2013 (link)) and subcloned into pLentiCRISPRv2-LoxP. HEK293LTV were cotransfected with the lentiviral plasmids pVSV-G (631530; Clontech) and psPAX2 (Vogel et al., 2015 (link)) and the viral supernatant directly used to infect HeLa cells. Transfected cells were subsequently selected with puromycin (P7255; Sigma-Aldrich). After selection, cells were transiently transfected with pCAG-Cre:GFP (13776; Addgene; Matsuda and Cepko, 2007 (link)) to remove the Cas9 and FACS sorted to enrich for the GFP-positive population. Limiting dilution then generated single cell clones. Finally, the KOs of lyspersin and LAMTOR1 (also referred to as LAMTOR1HM; de Araujo et al., 2017 (link)) were confirmed by PCR screen (Yu et al., 2014 (link)), Western blot analysis, and genotyping of the respective locus.

Filipek P.A., de Araujo M.E., Vogel G.F., De Smet C.H., Eberharter D., Rebsamen M., Rudashevskaya E.L., Kremser L., Yordanov T., Tschaikner P., Fürnrohr B.G., Lechner S., Dunzendorfer-Matt T., Scheffzek K., Bennett K.L., Superti-Furga G., Lindner H.H., Stasyk T, & Huber L.A. (2017). LAMTOR/Ragulator is a negative regulator of Arl8b- and BORC-dependent late endosomal positioning. The Journal of Cell Biology, 216(12), 4199-4215.

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Retroviral Vectors for Notch Signaling

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Retroviral vectors encoding Jagged1-IRES-EGFP and Delta-like1-IRES-EGFP were reported previously55 (link). The Jagged1 Slm mutant vector was generated by long PCR-based protocol using following primer pairs: 5′-AAG TGC ATC CCG CAC TCA GGA TGT GTC CAC GGT ACC TGC AAT GAA-3′ as a sense primer and 5′-TTC ATT GCA GGT ACC GTG GAC ACA TCC TGA GTG CGG GAT GCA CTT-3′ as an antisense primer. A cDNA encoding HA-NLG123 (link) was gift from Dr. Peter Scheiffele (University of Basel, Switzerland) and inserted into pMIGR156 (link). pCAG-Cre:GFP was purchased from Addgene (#13776). pCX-Myristoylated Venus57 (link) vector was gifted from Dr. Anna-Katerina Hadjantonakis (Sloan-Kettering Institute).

Hayashi Y., Nishimune H., Hozumi K., Saga Y., Harada A., Yuzaki M., Iwatsubo T., Kopan R, & Tomita T. (2016). A novel non-canonical Notch signaling regulates expression of synaptic vesicle proteins in excitatory neurons. Scientific Reports, 6, 23969.

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Generation of Sonic Hedgehog Mutant Mice

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The following strains were used: Shh^{tm1(EGFP/cre)Cjt}/J (Shh-EGFP-Cre) and B6.129S6-Shh^{tm2(cre/ERT2)Cjt}/J (Shh-Cre^ERT2) (Harfe et al., 2004 (link)); Bmpr1a^flox (Yuhki et al., 2004 (link)); and Gt(ROSA)26Sor^tm1(EYFP)Cos (R26R-YFP) (Srinivas et al., 2001 (link)). To generate Shh-null animals, Shh-EGFP-Cre mice were inter-crossed. To generate K79^tm2a mice, embryonic stem cells from clone EPD0179-4-A12 were purchased from KOMP and microinjected into blastocysts. Founder animals were crossed with E2a-Cre mice to remove the neomycin cassette. K79^tm2a mice were inter-crossed to generate K79-null mice. To generate K79-Cre mice, the 5,789 bp sequence upstream of the K79 transcriptional start site was PCR-amplified using BAC # RP24-152H23 and cloned into pCAG-Cre:GFP (Addgene #13776). Finally, the insert was purified and microinjected into (C57BL/6 × SJL) F2 mouse eggs and transferred into recipients. All studies were performed on mice of both genders in accordance with regulations established by the University of Michigan Unit for Laboratory Animal Medicine.

Mesler A.L., Veniaminova N.A., Lull M.V, & Wong S.Y. (2017). Hair follicle terminal differentiation is orchestrated by distinct early and late matrix progenitors. Cell reports, 19(4), 809-821.

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Construction and Characterization of Retrovector Derivatives

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All retrovector constructs were derived from the pPRIG retrovector or its relatives [40] (link). Two new pPRIG derivatives were used in this work: pPRiHy and pPRIZ vectors in which the eGFP cDNA in pPRIG was replaced by the Hygromycin- or Zeocin-resistance gene, respectively. mCAT1 cDNA was recovered from a lentiviral vector constructed by the Shinya Yamanaka’s lab (Kyoto University, Japan), and purchased from Addgene (pLenti UbC mSlc7a1, Plasmid #17224). mCAT1-HA cDNA was recovered from a pTarget mCAT-HA constructed by Yoshinao Kubo (Nagasaki University) provided by Sébastien Storck (INSERM U783, Paris, France). The Cre-encoding retrovector harbors a Cre-HA cDNA designed by modifying a Cre-GFP cDNA from Connie Cepko lab (Harvard Medical School, Boston, USA) and purchased from Addgene (pCAG Cre-GFP, Plasmid #13776).

Carlier G., Maugein A., Cordier C., Pechberty S., Garfa-Traoré M., Martin P., Scharfmann R, & Albagli O. (2014). Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation. PLoS ONE, 9(9), e108202.

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Generation of LRRK2 Knock-in iPS Cells

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A million of LRRK2 or 100550A iPS cells were harvested using Accutase (Sigma) and reverse-transfected with 1 μg of donor construct, 12 pmol spCas9 protein (Aldevron), and 18 pmol of sgRNA (LRRK2: 5′-GAACTCACATCTGAGGTCAG-3′, AARS1: 5′-GGGCGTATCGGACAGCTCGG-3′, Synthego), 4 μl P3000 reagent and later with 5 μl Lipofectamine 3000 (Thermo-Fisher). A mixture of transfection reagents was added onto a Cultrex-coated well first and then followed by resuspended LRRK2 iPS cells or 100550A iPS cells in fresh medium with 5 μM Y-27632 (Stemgent). Puromycin (500 ng/ml, Sigma) was added into the medium three to five days after transfection. Drug-resistant cells were replated at low density (5,000 cells/100 mm dish) and single cell colonies were manually selected afterwards. Clones with both 5’ and 3’ insertion positive genotyping results were further expanded and the Puromycin cassette was deleted by transient transfection of a CRE vector pCAG-Cre:GFP (Addgene #13776) and then plated at low density for single cell colonies. After a 2nd round of genotyping, positive clones were expanded and characterized.

Cai J., Kropf E., Hou Y.M, & Iacovitti L. (2021). A stress-free strategy to correct point mutations in patient iPS cells. Stem cell research, 53, 102332.

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Cre-mediated Cassette Excision

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For excision of the Puro-T2A-TK and of the TagRFP-Trap-Puro-T2A-TK cassettes from the genomes of correctly targeted clones, Cre recombinase was transiently expressed from pCAG-Cre:GFP (Addgene#13776), Ganciclovir (Invivogen, San Diego, USA) but was added to the medium 72 h following transfection for negative selection of cells that retained the TK.

Spraggon L., Martelotto L.G., Hmeljak J., Hitchman T.D., Wang J., Wang L., Slotkin E.K., Fan P.D., Reis-Filho J.S, & Ladanyi M. (2017). Generation of conditional oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing and homology-directed repair. The Journal of pathology, 242(1), 102-112.

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Knock-in EGFP at Mouse AVP Gene

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To knock-in EGFP at the mouse AVP gene, Xanthomonas TAL Nucleases (XTN; Transposagen Gene Editing Kit; Transposagen Biopharmaceuticals, Inc., Lexington, KY, USA) were designed to cause a mutation at the first exon of the vasopressin precursor gene around the initial ATG. For recombination, homology arms (5’: 0.9 kbp, 3’: 0.9 kbp) were amplified by polymerase chain reaction (PCR) from 129J mouse genomic DNA (from EB5 ES cells). The cDNA of enhanced GFP (EGFP; BD Biosciences) was fused into the first exon of the AVP. A PGK promoter-driven hygromycin-resistance selection cassette flanked by loxP sites was inserted downstream of EGFP. Vectors for the XTN pair with linearized targeting vector were co-transfected by electroporation (Mouse ES Cell Nucleofector™ kit and Nucleofector II; Lonza, Basel, Switzerland; Cat# VPH-1001) into EB5 ES cells. Homologous recombinant ES cells were selected with hygromycin and then screened by PCR. Targeted clones were confirmed by a PCR analysis with the 3’, 5’ and neo probes. The floxed PGK-hygro cassette was removed from subclones by transient transfection with Cre-expressing plasmid (pCAG-Cre:GFP; Addgene, #13776) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc. Cat# 11668030). The resultant subclones exhibited abilities to differentiate into hypothalamic neurons, including vasopressin.

Kawata M., Kodani Y., Ohkuma M., Miyachi E.I., Kaneko Y.S., Nakashima A., Suga H., Kameyama T., Saito K, & Nagasaki H. (2022). Long-range axonal projections of transplanted mouse embryonic stem cell-derived hypothalamic neurons into adult mouse brain. PLOS ONE, 17(11), e0276694.

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Targeted Deletion of miR-379/410 Cluster in ESCs

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sgRNAs (Mirg.up sgFwd/Rev and Mirg.dn sgFwd/Rev, Table S3) were designed to sites flanking the miR-379/410 cluster and were cloned into the pX458 plasmid (Addgene). HDR templates containing a loxP site flanked by 60 bp homology arms were designed to each sgRNA site and were synthesized as single-stranded oligonucleotides (IDT) (Mirg.up HDR template and Mirg.dn HDR template, Table S3). sgRNAs and HDR templates were co-transfected into rtTA;TetO-Ngn2-mCherry;Tubb3-GFP ESCs using Lipofectamine2000 (Thermo). Single cells were isolated by FACS into individual wells of a 96-well plate and screened for loxP insertion by PCR at the upstream and downstream insertion sites. A single cell clone with dual loxP insertion was confirmed to be maternal-specific by Sanger sequencing of allelic SNPs within loxP-containing PCR products. Next, the miR-379/410 cluster was deleted by transient transfection of pCAG-Cre:GFP (Addgene). Since cell clones were isolated by FACS for GFP-negative (Mirg^fl/+) and GFP-positive (Mirg^Δ/+) cells, and Cre-mediated deletion of miR-379/410 was confirmed by PCR.

Whipple A.J., Breton-Provencher V., Jacobs H.N., Chitta U.K., Sur M, & Sharp P.A. (2020). Imprinted maternally-expressed microRNAs antagonize paternally-driven gene programs in neurons. Molecular cell, 78(1), 85-95.e8.

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Rab7 Plasmid Constructs for Trafficking

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The plasmids used in this study are as follows:
Emerald-Rab7^WT (Addgene #54244, Davidson Lab), GFP-Rab7^WT (Addgene #12605, Pagano Lab), GFP-Rab7^Q67L (gift from Dr. James Casanova, University of Virginia), GFP-Rab7^T22N (Addgene #12660, Pagano Lab), pCAG-Cre-GFP (Addgene #13776, Cepko Lab), pCDNA3-2xHA-V1G1 (gift from Dr. Cecilia Bucci, University of Salento), pET-His-GST-tev-LIC (Addgene #29655, Gradia Lab), pGEX-4T-3-mR7BD (Addgene #79149, Edinger Lab), mCherry (Clontech), pmRFP-LC3 (Addgene #21075, Yoshimori Lab). To generate Emerald-Rab7^L8A, Rab7a with a point mutation at residue 8 from leucine to alanine (L8A) was cloned into pEmerald-C1 (Addgene#54734, Davidson Lab) at XhoI-BamHI sites by Genscript, using gene synthesis.

Mulligan R.J., Magaj M.M., Digilio L., Redemann S., Yap C.C, & Winckler B. (2024). Collapse of late endosomal pH elicits a rapid Rab7 response via V-ATPase and RILP. bioRxiv.

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Selective Activation of β-Arrestin Signaling

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The pcDNA3.1 vector carrying rM3Darr was provided by Professor Ken-ichiro Nakajima who generated this construct containing M3 with the R165L point mutation. AAV₉-hSyn-DIO-rM3Darr-mCherry was developed to selectively activate β-arrestin-biased signaling without perturbing G protein mediated pathways in response to CNO treatment as described previously^{36 (link)}. The Cre sequence from pCAG-Cre-GFP (Addgene Plasmid # 13776) was cloned into pAAV₂-THP-EGFP (Addgene Plasmid # 80336) through ECOR I and Sal I restriction enzyme sites. AAV₉-mCaMKIIα-EGFP-P2A-iCre, AAV₉-mCaMKIIα-EGFP, AAV₉-EF1α-Flex-ChrimsonR-tdTomato, AAV₉-THP-Cre, scAAV₁-hSyn-FlpO, and AAV₉-EF1α-fDIO-Cre-mCherry were packaged by Taitool Biological Co., Ltd. AAV₉-EF1α-DIO-eNpHR3.0-EYFP, AAV₉-EF1α-DIO-EYFP, AAV₉-hSyn-DIO-rM3Darr-mCherry, AAV₉-hSyn-DIO-mCherry, AAV₉-hSyn-NE2h-EGFP, and AAV₉-CAG-Cre were packaged by Neuron Biotech Co., Ltd. AAV₉-EF1α-DIO-hChR2(H134R)-mCherry and AAV₉-EF1α-DIO-mCherry were packaged from the BrainVTA Co., Ltd. AAV titers ranged from 2.0 × 10¹² to 2.5 × 10¹² vector genome (vg) ml⁻¹ were used in all experiments. High titer of scAAV₁-hSyn-FlpO (1 × 10¹³ vector genome (vg) ml⁻¹) was applied for anterograde infection.

Cheng D., Wu J., Yan E., Fan X., Wang F., Ma L, & Liu X. (2022). Noradrenergic consolidation of social recognition memory is mediated by β-arrestin–biased signaling in the mouse prefrontal cortex. Communications Biology, 5, 1097.

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