The Lamin B1 open reading frame (ORF) was subcloned from mCherry-LaminB1-10, a gift from M. Davidson (Addgene #55609), and the M.CviPI ORF with a C-terminal glycine-serine (GS) linker (GGSGGGS) was ordered as a gene block (Integrated DNA Technologies). An N-terminal FLAG tag and a nuclear localization signal (NLS) along with the M.CviPI ORF were then cloned N-terminal to Lamin B1 into pENTR (Invitrogen) using Gibson cloning (New England Biolabs, E2621X). The final overexpression construct for FLAG-NLS-M.CviPI-LaminB1 in pInducer20 (Invitrogen) was obtained through a recombination reaction using Gateway cloning (LR Clonase™ II, Thermo Fisher, 11-791-020) between the M.CviPI-pENTR construct and pInducer20. For the LIMe-ID experiments with EED and EZH2 inhibition, the M.CviPI-LaminB1 plasmid without the FLAG-NLS sequence was employed instead and cloned in an analogous manner.
Pinducer20
Manufactured by Thermo Fisher Scientific
The PInducer20 is a lab equipment product designed for controlled protein expression. It provides a regulated promoter system for inducible gene expression in a variety of host cells. The core function of the PInducer20 is to enable researchers to precisely control the timing and level of target protein production in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lr clonase 2, by Thermo Fisher Scientific (5 mentions)
Gene block, by Integrated DNA Technologies (4 mentions)
Gibson cloning, by New England Biolabs (4 mentions)
Pinducer20, by Addgene (2 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Ttrmpvir, by Addgene (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Quikchange site directed mutagenesis kit, by Thermo Fisher Scientific (1 mentions)
Gateway cloning, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Pdonr221, by Thermo Fisher Scientific (1 mentions)
8 protocols using pinducer20
1
Lamin B1 Fusion Protein Expression
2
FLAG-NLS-M.CviPI-LaminB1 Overexpression
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The Lamin B1 open reading frame (ORF) was subcloned from mCherry-LaminB1-10, a gift from M. Davidson (Addgene #55609), and the M.CviPI ORF with a C-terminal glycine-serine (GS) linker (GGSGGGS) was ordered as a gene block (Integrated DNA Technologies). An N-terminal FLAG tag and a nuclear localization signal (NLS) along with the M.CvIPI ORF were then cloned N-terminal to Lamin B1 into pENTR (Invitrogen) using Gibson cloning (New England Biolabs). The final overexpression construct for FLAG-NLS-M.CviPI-LaminB1 in pInducer20 (Invitrogen) was obtained through a recombination reaction using Gateway cloning (LR Clonase™ II, Thermo Fisher) between the M.CviPI-pENTR construct and pInducer20. For the LIMe-ID experiments with EED and EZH2 inhibition, the M.CviPI-LaminB1 plasmid without the FLAG-NLS sequence was employed instead and cloned in an analogous manner.
3
Microprotein Construct Generation
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pTUNAR ORF was synthesized (IDT technologies) fused with a flexible linker (GGGGSGGGGSGGGGS) and an HA tag epitope at the C-terminal part of the microprotein and flanked by EcoRI enzyme restrictions sites at both ends. After enzymatic digestion, constructs were ligated into the pENTR1A vector or the pMSCV vector. For the lentiviral vectors, the pTUNAR-HA tag construct was obtained by recombining donor vectors with the lentiviral inducible system pINDUCER20 (Invitrogen) or the pLV-CAG system using the Gateway Cloning Technology, following manufacturer’s instructions.
4
Cloning WT p53 into pInducer20 vector
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Using Gateway method, we cloned WT p53 (pCR8 p53 WT) into pInducer20 (Addgene) following the manufacturer’s instructions. Briefly, 50–150 ng of donor vector (pCR8 p53 WT) and 150 ng of pInducer20 plasmids were mixed with LR Clonase II (Cat# 11791, Invitrogen) and incubated at 25 °C for 1 h and reaction was terminated by adding Proteinase K. The reaction products were then used to transform DH5a bacteria and positive clones were selected on ampicillin containing agar plates80 (link)–84 (link).
5
Generation and Use of Inducible Viral Vectors
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Transfections and viral infections were performed as described (Timofeev et al, 2013 ). For production of lentiviruses, helper plasmids pMD2.g (Addgene plasmid #12259) and psPAX2 (Addgene plasmid #12260) were used. For Tet‐inducible gene knockdown with shRNAs, we used the retroviral vector TtRMPVIR (Addgene plasmid #27995), and for Tet‐inducible gene expression, we used the lentiviral vector pInducer20 (Addgene plasmid #44012). For expression of mutant p53, the cDNA for murine or human p53 was cloned into pInducer20 using the Gateway® System (Invitrogen). MEFs were immortalized by retroviral transduction with pMSCVhygro‐E1A.12S or pMSCVneo‐E1A.12S (Timofeev et al, 2013 ). For studies on Ras‐induced senescence, MEFs were transduced with MSCVhygro‐HRasG12V (Timofeev et al, 2013 ).
6
Generating Lentiviral Constructs with HA-Tagged pTINCR
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pTINCR ORF was synthesized (IDT technologies) fused with a flexible linker (GGGGSGGGGSGGGGS) and an HA-Tag epitope at the C-terminal or N-terminal part of the microprotein and flanked by EcoRI enzyme restrictions sites at both ends. After enzymatic digestion, constructs were ligated into the pENTR1A vector. pTINCR-SIM mut plasmid was purchased directly cloned into the pDONR201 plasmid, in-frame with an HA-tag epitope in its C-terminal (Proteogenix). Subsequently, pTINCR-HA, HA-pTINCR and pTINCR-SIMmut constructs (Supplementary Table 2 ) were obtained by recombining donor vectors with the lentiviral inducible system pINDUCER20 (Invitrogen) using the Gateway Cloning Technology, following manufacturer’s instructions. As specified, some experiments were reproduced using a pTINCR synthetic ORF (syORF), generated by mutating the pTINCR ORF in pTINCR-HA and pTINCR SIMmut constructs (Supplementary Table 2 ).
7
Inducible Expression of LCP1 Mutant
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Interleukin-3 (IL-3)–dependent murine myeloblast 32D cells (ATCC) were grown as described elsewhere.16 (link) The LCP1 human cDNA was obtained from GeneCopoeia. The missense mutation c.694 A>T (p.I232F) was created by using the QuikChange Site-Directed Mutagenesis Kit (Thermo Fisher Scientific). The wtLCP1 and LCP1 I232F constructs were cloned into the pInducer20 vector system by using Gateway cloning (Thermo Fisher Scientific). To induce expression, media were supplemented with 1 µg/mL of doxycycline (MilliporeSigma). doxycycline dose–response was performed to determine induction (supplemental Figure 1).
8
Overexpression of FLAP-MASTL Resistant to Silencing
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FLAP (3xFlag-GFP)-MASTL cDNA carrying silent mutations to make it MASTL resistant to MASTL siRNAs (siR) used in this study was cloned into the entry vector pDONR221 (ThermoFisher) and moved into the doxycycline inducible lentiviral destination vector pInducer20 (gift from Stephen Elledge, Addgene plasmid #44012) via gateway cloning. The resulting constructs were co-transfected with a vesicular stomatitis virus glycoprotein envelope expression (pVSV-G) and PAX2 (gift from Didier Trono, Addgene plasmid #12260) plasmids into viral production cells GP2-293 cells (gift from Lujambio lab) following standard protocols previously described.56 (link) Infectious supernatants were filtered, diluted 1:1 with complete medium containing 20 μg/mL polybrene, and applied to target cells for 24 h. Selection with neomycin (0.4 mg/mL) was initiated 48 h later and stable populations validated and used for subsequent experiments. Similar steps were followed to clone FLAP-MASTL-siR into a pQCXIN-FLAP vector24 (link) and to generate viral particles to infect target cells. Finally, pQCXIB histone H2B-mCherry retroviral vector24 (link) was co-transfected with pVSV-G into GP2-293 cells and virus particles collected as above to infect target cells followed by selection with Blasticidin (10 μg/mL).
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