Human circ‐CUX1 linear sequence (393 bp) was obtained from NB tissues by PCR (Appendix Table S2 ) and inserted into pLCDH‐ciR (Geenseed Biotech Co., Guangzhou, China). Human p200 CUX1 construct was provided by Dr. George Stratigopoulos, while p110 CUX1 was released by digestion or amplified using primers (Appendix Table S2 ), and subcloned into pcDNA3.1 (Invitrogen) or pCMV‐3Tag‐1A (Addgene, Cambridge, MA). Human EWSR1 cDNA (1,971 bp) and MAZ cDNA (1,482 bp) were provided by Dr. Ralf Janknecht or amplified from NB tissues with primers (Appendix Table S2 ), and their truncations were subcloned into pCMV‐N‐Myc or pCMV‐3Tag‐1A (Addgene). Mutation of circ‐CUX1 or EWSR1 was prepared with GeneTailor™ Site‐Directed Mutagenesis System (Invitrogen) and primers (Appendix Table S2 ). Oligonucleotides specific for shRNAs (Appendix Table S3 ) were inserted into GV298 (GeneChem Co., Ltd, Shanghai, China). Stable cells were screened by neomycin or puromycin (Invitrogen).
Pcmv n myc
Manufactured by Addgene
The PCMV-N-Myc is a plasmid vector that contains the N-terminal c-Myc tag sequence. It can be used for the expression and purification of recombinant proteins with an N-terminal c-Myc tag in mammalian cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Puromycin, by Thermo Fisher Scientific (4 mentions)
Pcdna3, by Thermo Fisher Scientific (3 mentions)
Neomycin, by Thermo Fisher Scientific (3 mentions)
Genetailor site directed mutagenesis system, by Thermo Fisher Scientific (3 mentions)
Gv298, by Genechem (3 mentions)
Pcmv 3tag 1c, by Addgene (3 mentions)
Dcas9 bfp krab, by Addgene (3 mentions)
Pcmv 3tag 1a, by Addgene (2 mentions)
Gv298 vector, by Genechem (2 mentions)
Pgex 6p 1, by Addgene (2 mentions)
Cv186, by Genechem (2 mentions)
Pet28a, by Addgene (1 mentions)
Cv186 lentivirus vector, by Genechem (1 mentions)
Dcas9 vpr, by Addgene (1 mentions)
Pegfp n1, by Addgene (1 mentions)
5 protocols using pcmv n myc
1
Constructing Neuroblastoma Molecular Constructs
2
Cloning and Manipulation of SPI1 and SPIB
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Human SPI1 cDNA (816 bp, Shanghai GeneChem Co., Ltd, China) were inserted into CV186 lentivirus vector (Genechem Co., Ltd). Human SPIB cDNA (789 bp) construct was provided by Dr. Zhe Liu.47 Their truncated fragments obtained using primer sets (Table S5 ) were inserted into pCMV‐N‐Myc, pCMV‐3Tag‐1C, pGEX‐6P‐1 or pET‐28a (Addgene, Watertown, MA). The shRNAs for SPI1 or SPIB were established by inserting oligonucleotides (Table S6 ) into GV298 vector (Shanghai GeneChem Co., Ltd), while small interfering RNAs (siRNAs) were synthesized (Table S6 ).
3
Cloning and Manipulation of Neuroblastoma Genes
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By using PCR, CNBP cDNA (540 bp) and its truncated forms were prepared from NB specimens (Table S8 ), and inserted into the CV186 (Shanghai Genechem Co., Ltd., China), pET28a‐mCherry (Genprice Inc., San Jose, CA, USA), pmCherry‐N1 (Genprice Inc.) or pCMV‐3Tag‐1A (Addgene, Cambridge, MA, USA). Human MYCN expression vector was a kind gift from Dr. Arturo Sala (College of Health, Medicine and Life Sciences, Brunel University London).54 PCR primers (Table S8 ) were used to amplify human SMARCC2 cDNA truncations (3645 bp) that were then subcloned into CV186 (Genechem Co., Ltd.), pCMV‐N‐MYC (Addgene) or pET28a‐EGFP (Genprice Inc.). Oligonucleotides specific for shRNAs against CNBP, MYCN, KPNB1, SMARCC2, SMARCC1 or SMARCA4 (Table S9 ) were ligated into GV298 vector (Genechem Co., Ltd). The dCas9‐BFP‐KRAB (Addgene) was used to produce single‐guide RNAs (sgRNAs; Table S9 ) against upstream or downstream regions of the transcription start sites for CNBP, BYSL, NOP58 or RRP9. Puromycin (Invitrogen) screening was undertaken for establishment of stable cells.
4
Engineered Transcription Factors and Vectors
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Human MZF1 coding sequence (CDS, 2205 bp), MZF1 cDNA (2920 bp), YY1 cDNA (1245 bp) and corresponding truncations were obtained from NB tissues by PCR primers (Table S2 ), and inserted into pcDNA3.1 (Invitrogen), pEGFP-N1, pCMV-3Tag-1C, pCMV-C-Flag, pCMV-N-MYC (Addgene, Cambridge, MA), or lentiviral expression vector CV186 (Genechem Co., Ltd, Shanghai, China), respectively. Human HK2 and PGK1 expression vectors were obtained from Genechem Co., Ltd. Mutation and frame-shift deletion of GFP or MZF1-uORF was prepared with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) and primers (Table S2 ). Oligonucleotides encoding short hairpin RNAs (shRNAs) specific for MZF1, HK2, PGK1, MZF1-uORF, or YY1 (Table S3 ) were subcloned into GV298 (Genechem Co., Ltd). Single guide RNAs (sgRNAs) were designed using Guide Design Resources (http://crispr.mit.edu ) to target upstream or downstream region relative to transcription start site of MZF1 (Table S2 ), and inserted into dCas9-VPR or dCas9-BFP-KRAB (Addgene), respectively. Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).
5
Molecular Cloning and Cell Line Generation
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Human HNF4A and MYCN expression vectors were provided by Dr. David Martinez Selva [16 (link)] and Dr. Arturo Sala [17 (link)], respectively. Human HNF4A-AS1 cDNA (648 bp), hnRNPU cDNA (2478 bp), CTCF cDNA (2184 bp), and their truncations were amplified from NB tissues (Additional file 1: Table S2 ) and subcloned into pcDNA3.1 (Invitrogen), pCMV-3Tag-1C, pCMV-N-Myc, and pGEX-6P-1 (Addgene, Cambridge, MA), respectively. Mutation of short hairpin RNA (shRNA)-targeting site of HNF4A-AS1 or hnRNPU RGG residues was performed with GeneTailorTM Site-Directed Mutagenesis System (Invitrogen) and primers (Additional file 1: Table S2 ). Oligonucleotides encoding shRNAs specific for HNF4A, HK2, SLC2A1, MYCN, HNF4A-AS1, hnRNPU, or CTCF (Additional file 1: Table S3 ) were subcloned into GV298 (Genechem Co., Ltd, Shanghai, China). Single guide RNAs (sgRNAs) targeting downstream region of HNF4A-AS1 transcription start site (Additional file 1: Table S3 ) were inserted into dCas9-BFP-KRAB (Addgene). Stable cell lines were screened by administration of neomycin or puromycin (Invitrogen).
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