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Pcalnl dsred

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The PCALNL-DsRed is a plasmid that expresses the DsRed fluorescent protein under the control of the CAG promoter. It is designed for the expression of DsRed in mammalian cells.

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Lab products found in correlation

Prl tk, by Promega (3 mentions) Pcag dsred, by Addgene (3 mentions) Pcag gfp, by Addgene (3 mentions) Pcag yfp, by Addgene (3 mentions) Pcag cfp, by Addgene (2 mentions) Pegfp tub, by Takara Bio (1 mentions) Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions) Amaxa mouse nucleofector kit, by Lonza (1 mentions) Pcag h2b tdirfp ip, by Addgene (1 mentions) Pcagen, by Addgene (1 mentions) Pcag cre, by Addgene (1 mentions) Pcalnl gfp, by Addgene (1 mentions) Pdire, by Addgene (1 mentions)

9 protocols using pcalnl dsred

1

Molecular Tools for Neuroscience Research

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BDNF-mCherry was previously used in (24 (link)) and ATAT1-GFP (27099), pCALNL-DsRed (13769), and pEGFP α-tubulin K40Q (105302) were purchased from Addgene (www.addgene.org/). pEGFP-Tub was obtained from Clontech (www.takarabio.com/). pCAG mEmerald-LAMP1 and pCAG mito-DsRED were provided by F. Polleux (Columbia University, New York, USA), and pCAG-iCreERT2 was provided by A. Tye (NIMR, UK). pCX-Cre plasmid was designed and provided by X. Morin (Institut de Biologie de l’Ecole Normale Superieure IBENS, France). ATAT1 truncation constructs, FLAG-ATAT1(amino acids 1 to 333), FLAG-ATAT1(amino acids 1 to 286), FLAG-ATAT1(amino acids 1 to 242), and FLAG-ATAT1(amino acids 1 to 196) were synthesized as gBlocks and inserted to a pCIG2 vector. The Atat1 short hairpin RNA (shRNA) sequence was 5′-GCAGCAAATCATGACTATTGT-3′ (30 (link)). Atat1 sequence was inserted in pBS/U6-ploxPneo plasmid (provided by X. Coumoul). Lis1 shRNA sequence was 5′-GAGATGAACTAAATCGAGCTA-3′ (38 (link)) and was subcloned in pCA-b-EGFPm5 silencer 3, a gift from M. Vermeren (King’s College London, UK). All construct sequences were verified by Sanger sequencing.
Even A., Morelli G., Broix L., Scaramuzzino C., Turchetto S., Gladwyn-Ng I., Le Bail R., Shilian M., Freeman S., Magiera M.M., Jijumon A.S., Krusy N., Malgrange B., Brone B., Dietrich P., Dragatsis I., Janke C., Saudou F., Weil M, & Nguyen L. (2019). ATAT1-enriched vesicles promote microtubule acetylation via axonal transport. Science Advances, 5(12), eaax2705.
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2

NPCs Nucleofection and Stable Lines

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NPCs were nucleofected with the pCALNL-dsRED plasmid (Addgene plasmid no. 13769) following the Amaxa mouse nucleofector kit recommendations (Lonza). Neomycin was added to select the nucleofected cells 48 hours after and was constantly kept in the NPC media. Transfection efficiency in these NPCs was checked by dsRED expression upon pCAG-CRE plasmid (Addgene plasmid no. 13775) nucleofection.
To tag cells with a constitutively expressed fluorescent protein before embryo injection, cells were either transfected with pCAG-H2B-tdiRFP-IP (Addgene plasmid no. 47884), containing puromycin resistance), or with PiggyBac vectors to express dsRED. A PiggyBac transposase-encoding plasmid and pCAG-dsRED-hygroR (PB-dsRED, hygromycin resistance) were cotransfected to randomly insert dsRED-hygroR in the genome. All transfections were performed using a Lipofectamine 3000 kit (Invitrogen). Cells were FACS-sorted to generate stable lines and kept under the respective drug selection.
Frade J., Nakagawa S., Cortes P., di Vicino U., Romo N., Lluis F, & Cosma M.P. (2019). Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development. Science Advances, 5(10), eaax4199.
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3

Versatile Fluorescent Protein Expression Plasmids

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pCAG-GFP (Addgene plasmid 11150) (Matsuda and Cepko, 2004 (link)). pCAG-YFP (Addgene plasmid 11180) (Matsuda and Cepko, 2004 (link)). pCAG-DsRed (Addgene plasmid 11151) (Matsuda and Cepko, 2004 (link)). pRho-GFP-IRES-AP (referred to as Rho-GFP) (Emerson and Cepko, 2011 (link)). pCAG-nlacZ (Cepko lab, Harvard Medical School) pCAGEN (Addgene plasmid 11160) (Matsuda and Cepko, 2004 (link)). pCALNL-DsRed (Addgene plasmid 13769) (Matsuda and Cepko, 2004 (link)). pCAFNF-DsRed (Addgene plasmid 13771). (Matsuda and Cepko, 2004 (link)). pCALNL-luc2 (Tang et al., 2015 (link)). pRL-TK (#E2241; Promega, Madison, WI).
Tang J.C., Drokhlyansky E., Etemad B., Rudolph S., Guo B., Wang S., Ellis E.G., Li J.Z, & Cepko C.L. (2016). Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies. eLife, 5, e15312.
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Diverse Fluorescent Protein Expression Vectors

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pCAG-GFP (Addgene plasmid 11150) 39 (link), pCAG-YFP (Addgene plasmid 11180) 39 (link), pCAG-CFP (Addgene plasmid 11179) 39 (link), pCAG-tdT (Cepko lab, Harvard Medical School), pCAG-mCherry (Cepko lab, Harvard Medical School), pCAG-DsRed (Addgene plasmid 11151) 39 (link), pRL-TK (Promega, #E2241), pRho-GFP-IRES-AP (referred to as Rho-GFP in main text) 40 (link), pCAG-nlacZ (Cepko lab, Harvard Medical School). pAAV-CAG-FLEX-tdT was a gift from Edward Boyden (Addgene plasmid #28306), pCALNL-DsRed (Addgene plasmid #13769) 41 (link).
Tang J.C., Rudolph S., Dhande O.S., Abraira V.E., Choi S., Lapan S., Drew I.R., Drokhlyansky E., Huberman A.D., Regehr W.G, & Cepko C.L. (2015). Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase. Nature neuroscience, 18(9), 1334-1341.
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5

In Utero Electroporation for Neuronal Labeling

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Plasmids were pCAG-GFP, pCALNL-GFP (LoxP-Stop-LoxP-GFP; floxed-GFP), pCALNL-DsRed (LoxP-Stop-LoxP-DsREd; floxed DsRed), and pCAG-Cre (Addgene plasmids #11150, #13770, #13769, and #13775 gifts from Connie Cepko)62 (link). CMV-synaptophysin-GFP plasmid was obtained from Addgene (plasmid #27235)63 (link). For IUE64 , timed pregnant mice were anesthetized with isoflurane/oxygen, and a solution containing a mixture of the specified plasmids at a concentration of 1 µg µl−1 each was injected into the embryo’s lateral ventricle using a 30 μm pulled glass micropipette. Five voltage pulses (38 mV, 50 ms) were applied using external paddles oriented in order to target S1, S2, or auditory cortex. After birth, pups were pups were anesthetized with carprofen (5 mg kg−1 bodyweight), or by induction of hypothermia, and fixed by intracardiac perfusion with ice-cold 4% paraformaldehy in PBS, pH 7.4, and at the specified developmental stages39 (link). Reconstructions of single neurons were performed by electroporating pCALNL-GFP (1 µg µl−1) and a low concentration of pCAG-Cre (20 ng µl−1) to label neurons sparsely64 .
De León Reyes N.S., Mederos S., Varela I., Weiss L.A., Perea G., Galazo M.J, & Nieto M. (2019). Transient callosal projections of L4 neurons are eliminated for the acquisition of local connectivity. Nature Communications, 10, 4549.
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6

Kozak-luc2 Insertion Into pCALNL-DsRed

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An EcoRI/NotI flanked and restriction digested fragment containing Kozak-luc2 was inserted into the backbone of EcoRI/NotI digested pCALNL-DsRed (Addgene plasmid #13769) 41 (link), replacing DsRed in the original vector.
Tang J.C., Rudolph S., Dhande O.S., Abraira V.E., Choi S., Lapan S., Drew I.R., Drokhlyansky E., Huberman A.D., Regehr W.G, & Cepko C.L. (2015). Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase. Nature neuroscience, 18(9), 1334-1341.
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7

Kozak-luc2 Insertion Into pCALNL-DsRed

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An EcoRI/NotI flanked and restriction digested fragment containing Kozak-luc2 was inserted into the backbone of EcoRI/NotI digested pCALNL-DsRed (Addgene plasmid #13769) 41 (link), replacing DsRed in the original vector.
Tang J.C., Rudolph S., Dhande O.S., Abraira V.E., Choi S., Lapan S., Drew I.R., Drokhlyansky E., Huberman A.D., Regehr W.G, & Cepko C.L. (2015). Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase. Nature neuroscience, 18(9), 1334-1341.
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8

Diverse Fluorescent Protein Expression Vectors

Cited 1 time
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pCAG-GFP (Addgene plasmid 11150) 39 (link), pCAG-YFP (Addgene plasmid 11180) 39 (link), pCAG-CFP (Addgene plasmid 11179) 39 (link), pCAG-tdT (Cepko lab, Harvard Medical School), pCAG-mCherry (Cepko lab, Harvard Medical School), pCAG-DsRed (Addgene plasmid 11151) 39 (link), pRL-TK (Promega, #E2241), pRho-GFP-IRES-AP (referred to as Rho-GFP in main text) 40 (link), pCAG-nlacZ (Cepko lab, Harvard Medical School). pAAV-CAG-FLEX-tdT was a gift from Edward Boyden (Addgene plasmid #28306), pCALNL-DsRed (Addgene plasmid #13769) 41 (link).
Tang J.C., Rudolph S., Dhande O.S., Abraira V.E., Choi S., Lapan S., Drew I.R., Drokhlyansky E., Huberman A.D., Regehr W.G, & Cepko C.L. (2015). Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase. Nature neuroscience, 18(9), 1334-1341.
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9

GBP-split Cre Constructs in Transgenic Mice

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Example 3

Many responder cassettes in transgenic mice are activated by the Cre recombinase. GBP-split Cre constructs (FIG. 9) were made as follows. Two split Cre pairs, 19T to 104L+106D to 343R, and 19T to 59N+60N to 343R, were fused to GBP1, 2, 4, 5, 6 and 7. A 12- or 15-amino acid linker was inserted between GBP and the split Cre fragment. Each split Cre fragment was amplified from pDIRE (Addgene plasmid 26745) (encoding a codon-optimized Cre, or iCre), adding AgeI-koz-NLS-NheI-MfeI on the 5′ end and NotI on the 3′ end. All fragments were cloned in place of GFP in the pCAG-GFP vector via AgeI/NotI. GBPs were subsequently cloned into these vectors via NheI/MfeI.

GBP Split Cre constructs were combined in many possible pairs and transfected into 293T cells along with pCALNLdsRed (Addgene plasmid 13769) (FIG. 10). pCAG-GFP or pBluescript were included for duplicate transfections for each split Cre pair.

US09949464B2. Target molecules for transcriptional control systems (2018-04-24). President and Fellows of Harvard College [US]. Inventors: Chung Yiu Jonathan Tang [US], Constance L. Cepko [US].
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