Full-length and truncated (N-terminal, ~28 kDa) mouse Cyr61 were amplified by PCR using the primers (for full-length cDNA: forward primer, 5′-AGATTCTAGAGCTAGCATGAGCTCCAGCACCTT-3′; reverse primer, 5′-CAGATCCTTGCGGCCGCTTAGTCCCTGAACTTGTG-3′; for truncated cDNA: forward primer, 5′-AGATTCTAGAGCTAGCATGAGCTCCAGCACCTT-3′; reverse primer, 5′-CAGATCCTTGCGGCCGCTTACTTGGAGCACTGGG-3′) from Cyr61 (NM_010516) Mouse Tagged ORF Clone (OriGene, MR221828) as previously reported (28 (link)), followed by subcloning into pCDH-CMV-MCS-EF1-copGFP lentivirus expression vector (System Biosciences, CD511B-1). To prepare the lentivirus, HEK293T cells (CRL-3126, ATCC) were cotransfected with lentiviral expression vectors and packaging vectors (System Biosciences) for 8 hours. The medium was replaced with fresh medium; the cultures were incubated for 48 hours, followed by collection of the medium supernatants containing lentivirus. MSCs were transduced with lentivirus in the presence of 8 μg/mL polybrene (MilliporeSigma).
Mouse tagged orf clone
Manufactured by OriGene
Sourced in United States
The Mouse Tagged ORF Clone is a laboratory tool that provides a purified and sequence-verified DNA clone expressing a specific open reading frame (ORF) from a mouse gene. It is designed to facilitate the study and characterization of mouse gene function.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (1 mentions)
Sgrna cloning vector, by Addgene (1 mentions)
Microinjector, by Eppendorf (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 empty vector, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pcmv6 ac gfp, by OriGene (1 mentions)
C terminal tgfp tag, by OriGene (1 mentions)
9 protocols using mouse tagged orf clone
1
Cyr61 Lentiviral Expression System
2
shRNA Suppression and Rescue of C11orf46
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Plasmids expressing interfering shRNA were generated to suppress endogenous C11orf46 protein expression utilizing the pSUPERIOR.puro vector system (Oligoengine). Their target sequences are: C11orf46 shRNA-1 with strong suppression; 5′-CAAACTGAATTTGCTCCAGAA- 3′ and C11orf46 shRNA-2 with milder suppression; 5′- GAAGACAGCTTGTACCTGGTT- 3′. A scrambled sequence that shows no homology to any known messenger RNA was utilized to produce the Control shRNA (5′-ATCTCGCTTGGGCGAGAGTAAG-3′). The HA and scFv-tagged RNAi resistant wild-type human C11orf46 expression constructs containing four silent mutations (underlined) in the target sequence of C11orf46 shRNA (5′- CAAACA GAG TTC GCA CCAGAA-3′) were produced and cloned into CAG promoter driven plasmid (pCAGGS1vector) for rescue experiments. dCas9-SunTag coding sequence was also transferred into pCAGGS1 vector. Two single guide RNA sequences each Dclk1 or Sema6a promoter region were using online CRISPR design tool67 (link). Those protospacer sequences were cloned into sgRNA cloning vector (Addgene #41824) with the protocol distributed on the addgene website (https//media.addgene.org/data/93/40/adf4a4fe-5e77-11e2-9c30-003048dd6500.pdf). The protospacer sequences are listed in Supplementary Table 5 . CAG promoter driven mouse Sema6a expression plasmid (CAG-mSema6a) was cloned using Sema6a (NM_018744) Mouse Tagged ORF clone (ORIGENE, Cat#MR211486).
3
Prkce cRNA Microinjection in Oocytes
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Microinjections were performed using a microinjector (Eppendorf). To obtain Prkce cRNA, Prkce (NM_011104) Mouse Tagged ORF Clone (OriGene) was linearized using Ase I (NEW ENGLAND Biolabs). HiScribe T7 ARCA mRNA kit (NEW ENGLAND Biolabs) was used to produce 5′ capped and 3′ polyA‐tailed mRNA, purified using an Invitrogen MEGAclear Transcription Clean‐Up Kit (ThermoFisher Scientific). The concentration of Prkce cRNA was determined using Nanodrop 2000 (ThermoFisher Scientific). The Prkce cRNA solution (671 ng/μl) or nuclease‐free water was microinjected into the cytoplasm of GV oocytes in an M2 medium drop supplemented with 2.5 μM milrinone. After microinjection, the oocytes were washed in M2 and matured in vitro.
4
Establishing Stable Cell Lines for FOXK2 and IRE1α Overexpression
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The FOXK2-expressing vector [FOXK2 cDNA ORF-pcDNA3.1/C(K)DYK, OHu30465, Genscript] was used for generating FOXK2-OE cell lines. The IRE1α-expressing vector was used for generating rescue overexpression in FOXK2-knockdown cells. The IRE1α-expressing vector was constructed by amplifying the full-length cDNA encoding IRE1α from the IRE1α-pcDNA3.EGFP plasmid (13009, Addgene) and ligating it into the pcDNA3.1 vector. Empty pcDNA3.1 vector (Invitrogen) was used as control. Sequences of plasmid were verified by Sanger sequencing. OC cells (OVCAR5 and OVCAR3) were transfected by using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, G418 sulfate was added to the complete culture medium (200 μg/mL for OVCAR5, 400 μg/mL for OVCAR3) to establish stably transfected cells. To rescue FOXK2 expression, cDNA encoding Foxk2 was amplified from the Foxk2 (NM_001080932) Mouse Tagged ORF Clone (ORIGENE) and subcloned into the pLenti-CMV vector. Empty pLenti-CMV vector was used as a control. HEK-293T cells were cotransfected with pLenti-Foxk2 and packaging mix using Lipofectamine 2000 reagent to generate lentiviral particles for transduction, as described above.
5
Constructing Stable COX-2 Cell Lines
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To construct stable COX-2-Overexpression cell lines, mouse Cox2 cDNA was obtained from Ptgs2 (NM_011198) Mouse Tagged ORF Clone (Clone ID MR227684, OriGene). The cDNA was cloned into the plasmid pLVX-IRES-ZsGreen1 to create the pLVX-COX-2-IRES-ZsGreen1 vector. Empty pLVX-IRES-ZsGreen1 was used as negative control. Recombinant construct with three helper plasmid pLP1, pLP2 and VSVG were transiently transfected into 293T cells using Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, United States) following the manufacturer’s instructions. After 48 h, the supernatant was collected and lentivirus particles were concentrated with 4 M NaCl and PEG6000. Then fresh virus was used to infect RAW264.7 cells with 10 ng/µl polybrene. GFP positive polyclone cells were sorted by a flow cytometer to generate stable cell lines. The polyclone cells were measured by western blotting.
To establish stable COX-2-Knockdown cell lines, two shRNA sequences were chosen for targeting COX-2 by NCBI BLAST (Tiedemann et al., 2012 (link)). The double-stranded oligonucleotides were annealed and inserted into plasmid pLL3.7 lentivirus vectors. The transfection and cell line sorting procedures were same as the ones used for construction of overexpression cell lines. Primer sequences in this study were listed inSupplementary Table 1 .
To establish stable COX-2-Knockdown cell lines, two shRNA sequences were chosen for targeting COX-2 by NCBI BLAST (Tiedemann et al., 2012 (link)). The double-stranded oligonucleotides were annealed and inserted into plasmid pLL3.7 lentivirus vectors. The transfection and cell line sorting procedures were same as the ones used for construction of overexpression cell lines. Primer sequences in this study were listed in
6
Cloning and Expression of Cldn6 and SFKs
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The coding region of mouse Cldn6 (full length, amino acids [AA] 1-219; Δ1/2C, AA 1-201; ΔC, AA 1-183) was amplified by RT-PCR using total RNA from F9 cells. A 3 × HA tag was added to the 5′ end of the Cldn6 sequence using a long-tail primer. The fragments were cloned into the XbaI/KpnI site of pD402 plasmid, in which the gene expression is driven by the PGK promoter. Mouse SFKs (Blk, Fgr, Frk, Fyn, Hck, Lck, Lyn, Src, and Yes1) were similarly amplified by PCR using cDNA of F9 cells or Blk Mouse Tagged ORF Clone (MR208004, OriGene, Rockville, MD, USA), and cloned into the EcoRI/SalI site of pFLAG-CMV-5.1 (E6908, Merck, Darmstadt, Germany). To produce the recombinant proteins, Blk-FLAG and Src-FLAG were subcloned into the KpnI/SacI site and the KpnI/HindIII site of plEx/Bac-4 (71726, Merck), while the C-terminus of Cldn6 (C185-V219) was inserted into the KpnI/HindIII site of plEx/Bac-4, which contains glutathione S-transferase (GST).
7
Transfection of NSCs with SOX21 Vector
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Lipofectamine 3000 reagent (Thermo Fisher Scientific, MA, USA) was used to transfect NSCs with mammalian vector pCMV6-AC-GFP containing SOX21 (NM_177753) Mouse Tagged ORF Clone (No: MG223510) or blank control pCMV6-AC-GFP (No: PS100010, OriGene Technologies, Inc. Rockville, MD, USA), according to the manufacturer’s instructions.
8
Tia1 Expression in Neural Stem Cells
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Mammalian vector pCMV6-AC-GFP, containing Tia1 (NM_009383) Mouse Tagged ORF Clone (No: MG226372, OriGene Technologies, Inc., Rockville, MD, USA) or blank control pCMV6-AC-GFP with C-terminal tGFP tag (No: PS100010, OriGene Technologies, Inc., Rockville, MD, USA), was transfected into NSCs using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer′ s instructions.
9
Functional Pharmacology of Od-DAR in HEK293 Cells
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For expression and functional pharmacological analyses of Od-DAR binding, the full-length cDNA of Od-DAR (long) was expressed in mammalian cells. The full-length cDNA was ordered from Gene Universal Inc. with the addition of a NheI (GCTAGC) site at the 5' end and a HindIII (AAGCTT) site at the 3' end. Initially, the gene was cloned into the pcDNA3.1(+)-EGFP vector. Later the full sequence of Od-DAR was subcloned into the pp2A-mCherry-N1 vector (a gift from Dorus Gadella, plasmid #84329, Addgene) using the NheI and HindIII enzymes. This vector enables the bicistronic expression of the protein of interest and mCherry. It was co-transfected with a mouse G-protein expressing vector (Gna15 (NM_010304) Mouse Tagged ORF Clone (MR205807, Origene) to permit signal transduction in HEK293 cells.
HEK293 cells were seeded into 24-well plates in Dulbecco's Modified Essential Media (DMEM) supplemented with 20mM HEPES and 10% heat inactivated fetal calf serum. The following day, cells were transfected using Lipofectamine 3000 Reagent (Invitrogen) according to manufacturer's instructions and allowed to grow overnight. Transfection with the Gna15 vector was the negative control, and co-transfection of Gna15 and a mouse ADR-expressing vector subcloned into the pp2A-mCherry-N1 vector was the positive control. Transfected cells were identified by mCherry expression prior to imaging.
HEK293 cells were seeded into 24-well plates in Dulbecco's Modified Essential Media (DMEM) supplemented with 20mM HEPES and 10% heat inactivated fetal calf serum. The following day, cells were transfected using Lipofectamine 3000 Reagent (Invitrogen) according to manufacturer's instructions and allowed to grow overnight. Transfection with the Gna15 vector was the negative control, and co-transfection of Gna15 and a mouse ADR-expressing vector subcloned into the pp2A-mCherry-N1 vector was the positive control. Transfected cells were identified by mCherry expression prior to imaging.
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