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Plx301

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The PLX301 is a laboratory equipment product offered by Addgene. It is designed for general laboratory use. The core function of the PLX301 is to provide a reliable and consistent platform for various laboratory tasks and experiments.

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Lab products found in correlation

Plx304, by Addgene (3 mentions) Lenticrispr v, by New England Biolabs (2 mentions) T4 polynucleotide kinase, by New England Biolabs (2 mentions) Quick ligase, by New England Biolabs (2 mentions) Gateway lr cloning, by Thermo Fisher Scientific (1 mentions) Plix403, by Addgene (1 mentions) Pslik neo, by Addgene (1 mentions) Pen ttmcs, by Addgene (1 mentions) Plko 1 vector, by Merck Group (1 mentions) Plx303, by Addgene (1 mentions) Recombinant il 2, by Thermo Fisher Scientific (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Pcmv vsv g, by Addgene (1 mentions) Ionomycin, by Merck Group (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Pspax2, by Addgene (1 mentions) Puromycin resistance, by Addgene (1 mentions) Gateway pdonr 221 vector, by Thermo Fisher Scientific (1 mentions) Endotoxin free kit 12362, by Qiagen (1 mentions) Pcw57, by Addgene (1 mentions)

Spelling variants of this product

PLX301 vector (4 citations)

10 protocols using plx301

1

Overexpression of Oncogenic RAS Variants

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KRAS(G12V), HRAS(G12V) and NRAS(G12V) were inserted in pLX301 (Addgene) using Gateway LR cloning (Thermo). Lentiviral particles were generated in HEK293T cells as described previously29 (link). A375 cells were transduced at a multiplicity of infection of 2 after which puromycin selection (2.5 μg mL−1) was applied for 72 hours. Resistant cells were maintained under puromycin selection until one passage before conducting experiments.
Posternak G., Tang X., Maisonneuve P., Jin T., Lavoie H., Daou S., Orlicky S., de Rugy T.G., Caldwell L., Chan K., Aman A., Prakesch M., Poda G., Mader P., Wong C., Maier S., Kitaygorodsky J., Larsen B., Colwill K., Yin Z., Ceccarelli D.F., Batey R.A., Taipale M., Kurinov I., Uehling D., Wrana J., Durocher D., Gingras A.C., Al-Awar R., Therrien M, & Sicheri F. (2020). Functional characterization of a PROTAC directed against BRAF mutant V600E. Nature chemical biology, 16(11), 1170-1178.
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2

Plasmid Construction for KANK1 and CXXC5 Studies

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The KANK1 cDNA without a stop codon in pENTR223 was purchased from DNASU (HsCD00516496). A full-length sequence of KANK1 with a stop codon was amplified by PCR using the purchased clone as a DNA template, and sub-cloned into pEN_TTmcs (Addgene #25755), pLX301, and pSLIK-Neo, which were purchased from Addgene (#25895 and #25735). pLIX405 was modified from pLIX403 (Addgene #41395) by replacing the V5 tag with EGFP. The pSlick-Neo-KANK1, pLIX405-KANK1, and pLX301-KANK1 were created using Gateway LR Clonase II. KANK1 shRNA V2LHS-50967 (KANK1-sh-1), KANK1 shRNA V2LHS-50970 (KANK1-sh-2), and a non-silencing (NS) control GIPZ plasmid (RHS4346) were purchased from GE Dharmacon. CXXC5 shRNA TRCN0000144558 (CXXC5-sh-1), TRCN0000142729 (CXXC5-sh-2), and a non-silencing control (NS) pLKO.1-vector were purchased from Sigma-Aldrich.
Cui Z., Shen Y., Chen K.H., Mittal S.K., Yang J.Y, & Zhang G. (2017). KANK1 inhibits cell growth by inducing apoptosis though regulating CXXC5 in human malignant peripheral nerve sheath tumors. Scientific Reports, 7, 40325.
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3

Overexpression of MEK and ETV1 in GIST cell lines

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cDNAs for human wild-type MEK1, wild-type MEK2, MEK1L115P mutant, MEK2L119P mutant were cloned into lentiviral based vector pLX301(Addgene). Lentivirus was produced in 293FT cells by standard methods using amphotropic packaging vector. GIST-T1 cells were infected and selected with 2ug/ml puromycin for 5 days at 48 hours post infection for subsequent biochemical and drug treatment studies.
To determine the effect of ETV1 overexpression on KIT transcript levels, cDNA of human ETV1 was cloned into MSCV-based retroviral vector pMIG (Addgene). Retrovirus was produced in 293FT cells by standard methods using amphotropic packaging vector.GIST882 and GIST-T1 cells were infected with empty vector or pMIG-ETV1. RNA was isolated 48 hours post infection to analyze KIT mRNA by qRTPCR.
Ran L., Sirota I., Cao Z., Murphy D., Chen Y., Shukla S., Xie Y., Kaufmann M.C., Gao D., Zhu S., Rossi F., Wongvipat J., Taguchi T., Tap W.D., Mellinghoff I.K., Besmer P., Antonescu C.R., Chen Y, & Chi P. (2015). Combined inhibition of MAP kinase and KIT signaling synergistically destabilizes ETV1 and suppresses GIST tumour growth. Cancer discovery, 5(3), 304-315.
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4

Generation of SF3B1 Variant Constructs

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WT and K700E FLAG-SF3B1 sequences were subcloned from Addgene
plasmids 82576 and 82577 into pDONR-A-HYG (Addgene 29635) to make
pENTR-SF3B1-WT and pENTR-SF3B1-K700E. Site-directed
mutagenesis with overlap extension PCR then created pENTR plasmids for the E592K, E622D,
K666R, K666N, R625H, K741N, and E902K variants of SF3B1, and these were
subcloned into lentiviral vector pLX301 (Addgene 25895). Plasmids used for
SF3B1 affi nity purification have been previously described17 (link). Site-directed mutagenesis of the
p3xFLAG-CMV-14-His6-FLAG-SF3B1 vector was done by overlap extension PCR to make the E592K
vector.
Choi I.Y., Ling J., Zhang J., Helmenstine E., Walter W., Bergman R., Philippe C., Manley J., Rouault-Pierre K., Li B., Wiseman D., Ouseph M., Bernard E., Li X., Haferlach T., Fazal S., Jain T., Gocke C., DeZern A, & Dalton W.B. (2023). The E592K variant of SF3B1 creates unique RNA missplicing and associates with high-risk MDS without ring sideroblasts. Research Square.
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5

Mitochondrial Protein Tagging and CRISPR

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In order to tag mitochondrial subunits (IDH2, UQCRB, UQCRC1, COX5B, and COX7A2L) with Venus or NanoLuc fragments, gene blocks were designed with gateway recombination cloning overhangs. The split Venus and NanoBiT sequences used in this study were adapted from by Ohashi et al. 32 (link) and Promega, respectively. IDT gBlocks were ligated into pDONR221 using Gateway BP Clonase II and subsequently shuttled into lentiviral expression vectors pLX301 (Addgene, #25895) and pLX304 (Addgene, #25890) using Gateway LR Clonase II according to manufacturer’s instructions. BiFC and NanoBiT construct pairs were sequentially transduced in U2OS cell lines to maximize transduction efficiency of both constructs. For CRISPR cloning, primer sequences were annealed and modified using T4 Polynucleotide Kinase (NEB), diluted, and ligated into lentiCRISPR v2 using Quick Ligase (NEB), following previous protocols 54 . For FAR1 overexpression, we used the lentiviral plasmid pLV[Exp]-Bsd-CMV>FAR1 (VB191206–1681rzb, VectorBuilder). Sequences for BiFC/NanoBiT constructs and CRISPR/Cas9 guides cloned into the lentiCRISPR v2 (puromycin, hygromycin selection) vectors are present in Supplementary Table 1.
Bennett C.F., O’Malley K.E., Perry E.A., Balsa E., Latorre-Muro P., Riley C.L., Luo C., Jedrychowski M., Gygi S.P, & Puigserver P. (2021). Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly. Nature chemical biology, 17(6), 703-710.
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6

Lentiviral Transduction of CL3EcoR Cells

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Lentiviruses expression constructs were used to stably transduce CL3EcoR cells. The ORF for each gene of interest was inserted into pLX301 or pLEX-MCS lentiviral vectors by gateway recombinational cloning or by DNA manipulation. Vectors pLX301 (catalogue number 25895) and pLEX-MCS (catalogue number OHS4735) were from Addgene and Thermo Scientific Open Biosystems respectively. Hygromycin resistant expression constructs for LIN9, LIN37, LIN52-S28A and LIN54 were provided by LL and JAD. Since CL3EcoR cells were hygromycin resistant, each of the inserts was sub-cloned into the puromycin resistant pLEX-MCS vector. Full length MYBL2 clone (HsCD00045539) and LIN9 (FL-LIN9) ORF, corresponding to a predicted LIN9 ORF 16 amino acids longer at the amino terminus than the original LIN9 were obtained from DNASU, a plasmid repository (https://dnasu.org/DNASU/). Gateway recombination cloning was used to insert these ORFs into the pLX301 destination vector. Both the constitutively active and the wild type form of FOXM1 were provided by Prof. Rene Medema (Netherlands Cancer Institute, Amsterdam, Netherlands) and sub-cloned into pLEX-MCS lentiviral vector. WT-LIN52-S28 and LIN52-S28E mutants were designed and ordered from GeneArt Gene Synthesis and Services [ThermoFisher Scientific (https://www.thermofisher.com/)]. The ORFs were cloned into pLEX-MCS vector.
Kumari R., Hummerich H., Shen X., Fischer M., Litovchick L., Mittnacht S., DeCaprio J.A, & Jat P.S. (2021). Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence. Scientific Reports, 11, 21506.
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7

Characterization of Immune Cell Phenotypes

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Jurkat cells were maintained in RPMI 1640 with 10% FBS. HeLa and 293T cells were maintained in DMEM with 10% FBS. Lentivirus was produced in 293T cells using pLX301 or pLX303 (Addgene #25895/25897), psPAX2, and pCMV-VSVG. PMA and ionomycin were obtained from Sigma-Aldrich. Recombinant IL-2 and IFN-γ were obtained from Peprotech. Antibodies were obtained from the following sources: PE- or Vio515-conjugated anti-IL-2 (N7.48 A) and PE-conjugated anti-IFN- γ (IFN-γ Secretion Assay-Detection Kit) from Miltenyi; APC-conjugated anti-CD45 (HI30), FITC-conjugated anti-HLA-A/B/C (W6/32), FITC-conjugated anti-HLA-DR (Tü36), FITC-conjugated anti-HLA-DP/DQ/DR (Tü39), PE-conjugated anti-HLA-DM (MaP.DM1), and PE-conjugated anti-TCR α/β (IP26) from BioLegend.
Lee M.N, & Meyerson M. (2021). Antigen identification for HLA class I- and II-restricted T cell receptors using cytokine-capturing antigen-presenting cells. Science immunology, 6(55), eabf4001.
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8

Mitochondrial Protein Tagging and CRISPR

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In order to tag mitochondrial subunits (IDH2, UQCRB, UQCRC1, COX5B, and COX7A2L) with Venus or NanoLuc fragments, gene blocks were designed with gateway recombination cloning overhangs. The split Venus and NanoBiT sequences used in this study were adapted from by Ohashi et al. 32 (link) and Promega, respectively. IDT gBlocks were ligated into pDONR221 using Gateway BP Clonase II and subsequently shuttled into lentiviral expression vectors pLX301 (Addgene, #25895) and pLX304 (Addgene, #25890) using Gateway LR Clonase II according to manufacturer’s instructions. BiFC and NanoBiT construct pairs were sequentially transduced in U2OS cell lines to maximize transduction efficiency of both constructs. For CRISPR cloning, primer sequences were annealed and modified using T4 Polynucleotide Kinase (NEB), diluted, and ligated into lentiCRISPR v2 using Quick Ligase (NEB), following previous protocols 54 . For FAR1 overexpression, we used the lentiviral plasmid pLV[Exp]-Bsd-CMV>FAR1 (VB191206–1681rzb, VectorBuilder). Sequences for BiFC/NanoBiT constructs and CRISPR/Cas9 guides cloned into the lentiCRISPR v2 (puromycin, hygromycin selection) vectors are present in Supplementary Table 1.
Bennett C.F., O’Malley K.E., Perry E.A., Balsa E., Latorre-Muro P., Riley C.L., Luo C., Jedrychowski M., Gygi S.P, & Puigserver P. (2021). Peroxisomal-derived ether phospholipids link nucleotides to respirasome assembly. Nature chemical biology, 17(6), 703-710.
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9

PARN and EGFP cDNA Cloning

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cDNAs encoding the PARN ORF (NM_002582; bases 147–2066) or EGFP were PCR amplified and cloned into the BsrGI and AgeI sites of pLX301 (puromycin resistance; Addgene, 25895) and pLX304 (blasticidin resistance; Addgene, 25890), gifts from D. Root.
Moon D.H., Segal M., Boyraz B., Guinan E., Hofmann I., Cahan P., Tai A.K, & Agarwal S. (2015). Poly(A)-specific ribonuclease (PARN) mediates 3′-end maturation of the telomerase RNA component. Nature genetics, 47(12), 1482-1488.
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10

Cloning and Overexpression of Alkbh5 and Alkbh5-HA

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Both mAlkbh5 and mAlkbh5-HAtag were amplified from the cDNA using gateway forward and reverse primer using PrimeSTAR GXL DNA Polymerase (Takahara Clontech # R050A-TAK), and the PCR product was purified using QIAquick PCR Purification Kit (Qiagen #28106), then shuttled to Gateway™ pDONR™221 Vector (Invitrogen#12536017) using Gateway™ BP Clonase™ II Enzyme mix (Invitrogen#11789020), then transformed to One Shot™ Stbl3™ Chemically Competent E. coli (Thermo Fisher #C737303), and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing. The correct clone was used as entry clone and then the cloned gene was shuttled to destination vector pLX301 (Addgene Plasmid #25895) For constitutive overexpression of either Alkbh5 or Alkbh5-HA, and pCW57.1 (Addgene Plasmid #41393) for periodic overexpression of either Alkbh5 or Alkbh5-HA using LR clonase (Thermo #11791020) based on manufacture protocol, and transformed to Stbl3 competent cells in case of Lentivirus destination vector, and positive clones were screen by colony PCR, and restriction digestion, and positive colonies were sent for sequencing, and confirmation , the positive colony was propagated and the plasmids were purified using Qiagen (Endotoxin free kit #12362), and used for making the virus.
Khodeer S., Klungland A., & Dahl J.A. (2021). ALKBH5 regulates somatic cell reprogramming in a phase specific manner.
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