The E7 sequences of A1, A4, and A5 were amplified using PCR with primers (forward, 5′-TTG CGG CCG CAC CAT GCA TGG AGA TAC ACC TAC ATT GC-3′; and reverse, 5′-TTG CGG CCG CTG GTT TCT GAG AAC AGA TGG GGC ACA C-3′) from clinical specimens, and cloned into the NotI site of p3xFLAG-CMV14 (Sigma-Aldrich, St. Louis, MO, USA) to fuse the 3xFLAG sequence to the C-terminus of the E7 protein. Then, the E7-FLAG sequence was amplified using PCR, and cloned between the PacI and XhoI sites of a retroviral transfer plasmid, pMXs-puro (Cell Biolabs, San Diego, CA, USA). A retrovirus vector expressing E7-FLAG was prepared via transfection of the transfer plasmid into GP2-293 packaging cells together with an envelope expression plasmid, pE-ampho (Takara). Human cervical keratinocytes immortalized with telomerase reverse transcriptase [36 (link)] were infected with the recombinant retrovirus expressing E7-FLAG, and selected with 1 μg/mL puromycin for 72 h, followed by culture without the drug for 48 h. The surviving cells were pooled and harvested for western blot analyses.
Pmxs puro
Manufactured by Cell Biolabs
Sourced in United States
The PMXs-puro is a plasmid vector that enables the expression of a puromycin resistance gene. It can be used for the selection of transfected mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
Fugene 6, by Promega (3 mentions)
Polybrene, by Merck Group (3 mentions)
Tr 1003 g, by Merck Group (2 mentions)
P3xflag cmv 14, by Merck Group (1 mentions)
Pe ampho, by Takara Bio (1 mentions)
Phusion hf polymerase, by Thermo Fisher Scientific (1 mentions)
Pretrox tre3g, by Thermo Fisher Scientific (1 mentions)
Pretrox tre3g, by Takara Bio (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
10 protocols using pmxs puro
1
Overexpressing E7 Protein in Human Cells
2
Generating Retroviral Expression Constructs
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Plasmids encoding human MAVS variants, hRIG-I(1–250), NS3/4A variants, and the ATeam1.03 were described previously13 (link), 16 (link), 20 (link), 34 (link). Plasmid encoding rat OPA1 was a gift from Naotada Ishihara (Kurume University, Japan). To generate the retroviral expression constructs, each cDNA was recloned into the retroviral vector pMXs-Puro (Cell Biolabs, San Diego, CA). The retroviral expression vectors were then transfected into the platinum packaging cell lines (Cell Biolabs), and the retroviral supernatant was harvested 48 h post-transfection and used to infect cells.
3
Retroviral Gene Delivery in Myoblasts
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Retroviral expression vector pMXs-Puro (Cell Biolabs, # RTV-012) was used for gene cloning and expression in human and mouse myoblasts. The open reading frames for red fluorescent protein Cherry and mouse Dll1 were cloned into pMXs-Puro vector by In-Fusion cloning. The identities of the DNA inserts in the plasmids were Sanger sequencing verified. MyoD-pCLBabe plasmid [82 (link)] was a gift from Stephen Tapscott (Addgene plasmid # 20917). Myoblasts were labelled by retroviral infection that delivers the pMXs-Cherry expression vector. MyoDER expression and chemical treatments were performed as previously reported [49 (link)].
4
MYOD1-Knockout Myoblasts Rescue by Ciona Factors
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Human MYOD1-knockout myoblasts were generated by CRISPR-Cas9 mediated gene editing and cultured as described previously (Zhang et al., 2020 (link)). Retroviral expression vector pMXs-Puro (Cell Biolabs, RTV-012) was used for cloning and the expression of the human MYOD1, Ciona MRF (Transcript Variant 2), and Ciona Ebf. The DNA sequences were verified by Sanger sequencing. For the myogenic rescue experiments, the sgRNA-insensitive version of human MYOD1 open reading frame was used. Retrovirus was produced through transfection of HEK293 cells using FuGENE 6 (Promega, E2692). Two days after transfection, virus medium was collected, filtered and used to infect human myoblasts assisted by polybrene (Sigma-Aldrich, TR-1003-G). When the culture reached 80–90% confluency, cells were induced for myogenic differentiation by switching to myoblast differentiation medium (2% horse serum in DMEM with 1% penicillin/streptomycin). Human myoblasts were differentiated for three days and used for immunostaining and RNA extraction. For immunostaining, the primary antibody for Myosin (Developmental Studies Hybridoma Bank, MF20) and the primary antibody for Myogenin (Developmental Studies Hybridoma Bank, F5D) were used. The qPCR primers for measurements of human MYMK and 18S expression are provided in the Supplemental Sequences File .
5
Retroviral Expression of Myogenic Factors
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Retroviral expression vector pMXs-Puro (Cell Biolabs, RTV-012) was used for cloning and expressing MymX and MymK orthologs. ORF inserts were codon-optimized and synthesized by Integrated DNA Technologies. The DNA sequences were verified by Sanger sequencing. For rescue experiments, the sgRNA-insensitive DNA cassettes were used. pLOVE-GFP plasmid was a gift from M. Ramalho-Santos (Addgene, plasmid no. 15949) (42 (link)). pMXs-Cherry plasmid was generated and described previously. Membrane targeting GFP was cloned from Addgene (plasmid no. 17787).
To produce retrovirus, retroviral plasmid was transfected to human embryonic kidney 293 cells using FuGENE 6 (Promega, E2692). Two days after transfection, viral medium was collected, filtered, and used to infect cells assisted by polybrene (Sigma-Aldrich, TR-1003-G). One day after viral infection, cells were switched to growth medium. To induce myogenic differentiation, cells were switched to myoblast differentiation medium (2% horse serum in DMEM with 1% penicillin/streptomycin). Human myoblasts can be fully differentiated 3 days after switching to differentiation medium. Mouse and lizard myoblasts were differentiated by switching to differentiation medium for at least 7 and 9 days, respectively.
To produce retrovirus, retroviral plasmid was transfected to human embryonic kidney 293 cells using FuGENE 6 (Promega, E2692). Two days after transfection, viral medium was collected, filtered, and used to infect cells assisted by polybrene (Sigma-Aldrich, TR-1003-G). One day after viral infection, cells were switched to growth medium. To induce myogenic differentiation, cells were switched to myoblast differentiation medium (2% horse serum in DMEM with 1% penicillin/streptomycin). Human myoblasts can be fully differentiated 3 days after switching to differentiation medium. Mouse and lizard myoblasts were differentiated by switching to differentiation medium for at least 7 and 9 days, respectively.
6
Genetic Constructs for Inducible NLRP3 Expression
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For inducible expression upon retroviral transduction, all constructs were inserted into the pRETROX Tre3G plasmid (Clontech). The mouse Nlrp3 encoding synthetic gene was ordered from GeneArt (Life Technologies). Truncated mouse Nlrp3 variants were prepared with PCR using Phusion HF polymerase (Thermo Fisher Scientific) using primers (Supplementary Table 2 : O1–O22, O61) and inserted into pRETRO X Tre3G at BamHI/EcoRI sites. Human NLRP3 and NLRC4 genes were obtained from Invivogen. Truncation human NLRP3 mutants were prepared with PCR using Phusion HF polymerase (primers: O23–O26) and inserted into pRETRO X Tre3G at the BamHI/NotI sites. Point mutations were introduced by site-directed mutagenesis using the O45–O60 primers and the Phusion HF polymerase. Chimeric constructs of mouse NLRP3 and human NLRC4 or human ASC (primers: O34–O44) and BRET constructs (primers: O28–O33) were prepared with PCR ligation. BRET constructs were further subcloned into pcDNA3 using the BamHI/NotI sites. pMXs-puro(Cell Biolabs)/ASC-GFP was used for control transduction of Asc−/− iBMDMs. All constructs were verified with DNA sequencing (GATC). The plasmid encoding hASC was a kind gift from K.A. Fitzgerald76 (link).
7
Cloning Net39 into pMXs-puro Vectors
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The ORF for Net39 was obtained in pCMV6-Entry backbone (Origene, MR203615). Net39 ORF was subcloned into the following custom-made pMXs-puro (Cell Biolabs, RTV-012) backbones: pMXs-puro-3xFLAG-HA (C-terminal) and pMXs-puro-miniTurbo (C-terminal). pMXs-puro-miniTurbo was generated by conventional cloning using pcDNA-V5-miniTurbo-NES (Addgene, #107170) as the PCR template.
8
Overexpression and Silencing of ATF4 in MIN6 Cells
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HA-Gsk-3βKM, Flag-DN-ATF4, and HA-delta F-βTrCP were sub cloned into the retroviral vector pMXs-puro (Cell Biolabs, San Diego, CA, USA). Two sequences used for the retroviral-mediated shRNA expression vector (pSirenQ; Invitrogen) targeting ATF4 were 5′-AAGCCTGACTCTGCTGCTTAC-3′ and 5′-AACCATGCCAGATGAGCTC-3′. Synthetic complementary single-stranded oligonucleotide DNA (Supplementary Table S1 ) was annealed to generate double-stranded DNAs of the target sequences. Annealed DNA was then inserted into the vector. The retroviruses were produced in HEK293T cells. MIN6 cells were transduced for 4 h with the virus and polybrene (Sigma-Aldrich) at 8 μg/mL. Following infection of MIN6 cells with the retrovirus, cells were selected by incubation with puromycin (2 days, 0.5 μg/mL). The cells were allowed to recover for 3 days before collection or stress treatment.
9
LEMD2 ORF Cloning and Mutagenesis
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The human open reading frame (ORF) of LEMD2 was purchased in pMGF196 from Addgene (97005). Subsequently, the LEMD2 ORF was subcloned into the retroviral vector pMXs-puro (Cell Biolabs, RTV-012). To obtain the c.T38>G mutation, we used the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, 200521).
10
Retroviral Expression of Myogenic Factors
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Retroviral expression vector pMXs-Puro (Cell Biolabs, RTV-012) was used for cloning and expressing MymX and MymK orthologs. Open reading frame (ORF) inserts were codon optimized and synthesized by IDT. The DNA sequences were verified by Sanger sequencing. For rescue experiments, the sgRNA insensitive DNA cassettes were used. MyoD-pCLBabe was a gift from Stephen Tapscott (Addgene plasmid # 20917) 13 . pLOVE-GFP plasmid was a gift from Miguel Ramalho-Santos (Addgene plasmid # 15949) 14 . pMXs-Cherry plasmid was generated and described previously 1 .
To produce retrovirus, retroviral plasmid was transfected to HEK293 cells using FuGENE 6 (Promega, E2692). Two days after transfection, viral medium was collected, filtered and used to infect cells assisted by polybrene (Sigma-Aldrich, TR-1003-G). One day after viral infection, cells were switched to growth medium. To induce myogenic differentiation, cells were switched to myoblast differentiation medium (2% horse serum in DMEM with 1% penicillin/streptomycin). Human myoblasts can be fully differentiated three days after switching to differentiation medium. Mouse and lizard myoblasts were differentiated by switching to differentiation medium for at least seven and nine days, respectively.
To produce retrovirus, retroviral plasmid was transfected to HEK293 cells using FuGENE 6 (Promega, E2692). Two days after transfection, viral medium was collected, filtered and used to infect cells assisted by polybrene (Sigma-Aldrich, TR-1003-G). One day after viral infection, cells were switched to growth medium. To induce myogenic differentiation, cells were switched to myoblast differentiation medium (2% horse serum in DMEM with 1% penicillin/streptomycin). Human myoblasts can be fully differentiated three days after switching to differentiation medium. Mouse and lizard myoblasts were differentiated by switching to differentiation medium for at least seven and nine days, respectively.
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