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Pcagen

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The PCAGEN is a laboratory equipment product used for the amplification of DNA sequences. It functions as a thermal cycler, enabling the controlled thermal cycling necessary for the polymerase chain reaction (PCR) process.

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Lab products found in correlation

Pcag gfp, by Addgene (2 mentions) Px330 u6 chimeric bb cbh hspcas9, by Addgene (2 mentions) Xhoi, by New England Biolabs (1 mentions) Pcag dsred, by Addgene (1 mentions) Pcalnl dsred, by Addgene (1 mentions) Pcag yfp, by Addgene (1 mentions) Prl tk, by Promega (1 mentions) Pcag ires gfp, by Addgene (1 mentions) Fast green, by Merck Group (1 mentions) Pegfp c3, by Takara Bio (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

PCAGEN vector (9 citations) PCAGEN plasmid (2 citations) PCAGEN-SBP-DICER1 (2 citations)

9 protocols using pcagen

1

Netrin-4 Electroporation Protocol

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DM electroporations were performed as described previously.39 (link),111 (link) Endodermal electroporations were performed with a similar method, but with the electroporation mix (plasmid of interest and/or pCAG-GFP, 1X PBS, 1X Fast Green, 1mM MgCl2, and 0.17% carboxymethylcellulose) injected into the empty space beneath the ventral side of the embryo while the negative electrode was held in place there. The positive electrode was placed directly above the negative electrode, centered along the neural tube, before the pulse was applied. pCAGEN-Ntn4 expression plasmid was constructed by cloning the full-length coding sequence out of mouse Ntn4-AP-His plasmid (Addgene 71980) using the primers in Table 1, and cloning into pCAGEN (Addgene 11160) with XhoI (NEB R0146) and NotI (NEB R0189). A second Ntn4 construct was also used, using a mouse Ntn4 (generously provided by Raphael Reuten) and cloned into the pMES vector,112 (link) with similar results.
Demler C., Lawlor J.C., Yelin R., Llivichuzcha-Loja D., Shaulov L., Kim D., Stewart M., Lee F., Schultheiss T, & Kurpios N. (2023). An atypical basement membrane forms a midline barrier in left-right asymmetric gut development. bioRxiv.
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2

Versatile Fluorescent Protein Expression Plasmids

Cited 2 times
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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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3

In Utero Electroporation Procedure

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In utero electroporation was performed as described (Shariati et al., 2013 (link)). Pregnant mice were anaesthetized by intramuscular injections of 88 mg ketamine and 132 mg xylazine per gram of body weight. The uterine horns were exposed and the plasmids mixed with Fast Green (Sigma) were microinjected in the lateral ventricles of E14.5 embryos. Five current pulses (50 milliseconds pulse/950 milliseconds interval) were delivered across the head of the embryos (36 V) targeting the dorsal-medial part of the cortex. An equal amount of pCAG-ires-GFP (0.5 µg/µl) was electroporated in all conditions to ensure an equal visualization of neuronal morphology. Plamids: pCAG-ires-GFP (Add Gene, 11,159); pCMV-GFP-ires-Cre (Fazzari et al., 2010 (link)); Nrg1-ICD was subcloned from pcDNA3.1 (see above) to pCAGEN (11160; Add Gene). All animal experiments were approved by the Ethics Committee of the KU Leuven.
Fazzari P., Snellinx A., Sabanov V., Ahmed T., Serneels L., Gartner A., Shariati S.A., Balschun D, & De Strooper B. (2014). Cell autonomous regulation of hippocampal circuitry via Aph1b-γ-secretase/neuregulin 1 signalling. eLife, 3, e02196.
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4

Generation of Plasmids for ACE2 and ADAM17 Studies

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Expression plasmids for human ACE2 (hACE2/pcDNA3.1) and mouse ACE2 (mACE2/pcDNA3.1) were previously described (16 (link)). A plasmid for a fusion protein of human ACE2 and green fluorescent protein (GFP) was from Origene. A plasmid for mouse ADAM17 (pAd17) was purchased from Addgene (catalog number 19141) (18 (link)). A plasmid for the E406A mutant of ADAM17 (pAd17E406A) was generated by in vitro mutagenesis changing a GAA to a GCA codon. The plasmid pACE2-Tom for CAG promoter-driven coexpression of mACE2 and tdTomato was generated based on pCAGEN (plasmid number 11160) from Addgene. Additional SacI and MluI restriction sites were inserted into the multicloning site. The mouse ACE2 coding sequence was amplified from mACE2/pcDNA3.1 and cloned between SacI and MluI restriction sites. The tdTomato coding sequence preceded by an internal ribosome entry site was amplified from LeGO-iT2 (Addgene, plasmid number 27343) and cloned between MluI and NotI restriction sites. Other plasmids used were pcDNA3.1(−) (Invitrogen/Life Technologies) and pEGFP-C3 (CLONTECH) for cytomegalovirus (CMV) promoter-driven expression of enhanced GFP.
Pedersen K.B., Chodavarapu H., Porretta C., Robinson L.K, & Lazartigues E. (2015). Dynamics of ADAM17-Mediated Shedding of ACE2 Applied to Pancreatic Islets of Male db/db Mice. Endocrinology, 156(12), 4411-4425.
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5

Cloning and Construction of ZIKV and TBK1 Plasmids

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ZIKV NS5, MT and RdRp domains were amplified with PCR from ZIKV MR766 strain (GenBank Accession Number: AY632535.2) and then cloned into the vector pCAGEN (Addgene plasmid number 11160) with FLAG, hemagglutinin (HA) or Myc tag at the N terminus. Human TBK1 was amplified and then cloned to pCAGEN with an HA or FLAG tag at the N terminus. TBK1 truncations were constructed into pCAGENHA or FLAG vector. TBK1 S347Amutation was constructed into pCAGEN-FLAG vector. All primers used for plasmid construction are listed in Table 1. All in-house-constructed plasmids were subjected to DNA sequencing. The construction of Myc-RIG-I (N-terminal domain) and Myc-MAVS (Nan et al., 2014 (link)) were described previously.
Lin S., Yang S., He J., Guest J.D., Ma Z., Yang L., Pierce B.G., Tang Q, & Zhang Y.J. (2018). Zika virus NS5 protein antagonizes type I interferon production via blocking TBK1 activation. Virology, 527, 180-187.
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6

Genetic Modification in Organoids

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For overexpression constructs, based on the Sleeping Beauty Transposase System, the CMV promoter from pCMV(CAT)T7-SB100 (Addgene cat. No.: 34879)52 (link) was replaced with CAG promotor from pCAGEN (Addgene cat. No.: 11160)53 (link). IRDR-R and IRDR-L sequences from pT2/LTR7-GFP (Addgene cat. No.: 62541)54 (link) were cloned into pCAGEN to produce pCAG-GS/IR. cDNAs used for overexpression were amplified from human cDNA and cloned into the MCS of pCAG-GS/IR. With the help of sleeping beauty transposase SB100X (pCAG-SB100X), CAG-GFP and CAG-oncogenes were integrated into the genome of cells in organoids. To introduce gene mutations, short guide RNAs of tumour suppressors were cloned into CRISPR/Cas9 vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene cat. No.: 42230)55 (link). All cloning primers are listed in the Supplementary Table 2 and 3.
Bian S., Repic M., Guo Z., Kavirayani A., Burkard T., Bagley J.A., Krauditsch C, & Knoblich J.A. (2018). Genetically engineered cerebral organoids model brain tumour formation. Nature methods, 15(8), 631-639.
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7

Cloning HA-tagged TOMT from Mouse Cochlea

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Mouse cochlear cDNA was used as a PCR template to amplify Tomt coding sequence (based on NCBI Consensus Coding Sequence # CCDS40044.1) using PCR primers containing HA tag, EcoRI, and NotI. HA-Tomt was ligated into pCAGEN (Addgene #11160) digested with EcoRI and NotI. PCR primers were: (i) 5’-AAAAAAAAAAGAATTCGCCACCATGTACCCATACGATGTTCCAGATTACGCTTCCCCTGCCATTGCACT-3’ containing EcoRI sequence, Kozak sequence and HA tag; (ii) 5’-AAAAAAAAAAGCGGCCGCTCAGCCGGGTCCAGTATAGG-3’ containing NotI sequence.
Cunningham C.L., Wu Z., Jafari A., Zhao B., Schrode K., Harkins-Perry S., Lauer A, & Müller U. (2017). The murine catecholamine methyltransferase mTOMT is essential for mechanotransduction by cochlear hair cells. eLife, 6, e24318.
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8

In utero Electroporation of Mouse Cortex

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In utero electroporation was performed as previously described with slight modifications 22. Briefly, pregnant wild‐type B6 mice were exposed to 1.8% isoflurane in oxygen during surgery and the embryos at embryonic day 15 were used for in vivo electroporation of the cortex. Control plasmid pCAGEN (Addgene) or pCAG‐Myc‐DISC1 was mixed with pCAG‐GFP (Addgene) in a 3:1 ratio and injected into one of the lateral ventricles of embryo brain using a glass micropipette. The embryonic brain then received five pulses (30 V for 50 ms) of electric shock with 950‐ms intervals using an ECM830 square wave pulse generator (BTX, Harvard Apparatus). Poly(I:C) injection, perfusion, brain fixation/sectioning, and neuronal image acquisition were performed as described in the previous sections.
Chen C., Liu H, & Hsueh Y. (2016). TLR3 downregulates expression of schizophrenia gene Disc1 via MYD88 to control neuronal morphology. EMBO Reports, 18(1), 169-183.
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9

Genetic Modification in Organoids

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For overexpression constructs, based on the Sleeping Beauty Transposase System, the CMV promoter from pCMV(CAT)T7-SB100 (Addgene cat. No.: 34879)52 (link) was replaced with CAG promotor from pCAGEN (Addgene cat. No.: 11160)53 (link). IRDR-R and IRDR-L sequences from pT2/LTR7-GFP (Addgene cat. No.: 62541)54 (link) were cloned into pCAGEN to produce pCAG-GS/IR. cDNAs used for overexpression were amplified from human cDNA and cloned into the MCS of pCAG-GS/IR. With the help of sleeping beauty transposase SB100X (pCAG-SB100X), CAG-GFP and CAG-oncogenes were integrated into the genome of cells in organoids. To introduce gene mutations, short guide RNAs of tumour suppressors were cloned into CRISPR/Cas9 vector pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene cat. No.: 42230)55 (link). All cloning primers are listed in the Supplementary Table 2 and 3.
Bian S., Repic M., Guo Z., Kavirayani A., Burkard T., Bagley J.A., Krauditsch C, & Knoblich J.A. (2018). Genetically engineered cerebral organoids model brain tumour formation. Nature methods, 15(8), 631-639.
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