Untagged Dll1 and Dll4 ORFs were PCR-amplified with primer pairs Dll1-for (SpeI) ACT AGT GCC ACC ATG TCT TAC GGT CAA GGG TCC AGC / Dll1-rev (AseI) GAT CAT TAA TTC ACA CCT CAG TCG CTA TAA CAC ACT CAT CCT TTT C and Dll4-for (NheI) GCT AGC AAT TCA TGA CGC CTG CGT CCC G / Dll4-rev (NdeI) CAT ATG TTA TAC CTC TGT GGC AAT CAC. Restriction sites introduced via primers were used to insert PCR products into NheI-NdeI sites of a shuttle vector containing IRES-GFP. Dll-IRES-GFP constructs were then subcloned into pMP8.CAG-Stop using restriction enzymes SwaI and MluI. HA-tagged versions of the above constructs for quantification of protein levels (Fig 1D ) were cloned in a similar way: PCR primers for Dll1-HA were Dll1-for (SpeI; see above) / Dll1-HA-rev (AseI) ATT AAT CTA AGC GTA ATC TGG AAC ATC GTA TGG GTA CAT ACT AGA CAC CTC AGT CGC TAT AAC ACA C. A C-terminal HA-tag was introduced to the Dll4 ORF via gene synthesis (Life technologies) of the flanking regions of Dll4-HA lacking the central AflII/EcoRV Dll4 fragment that was cloned into the synthesised fragment; the complete Dll4-HA was cloned into NheI/NdeI of the shuttle vector. HA-tagged constructs were Cre-recombined in bacteria of the recombination strain SW106 (NCI at Frederick).
Gene synthesis
Manufactured by Thermo Fisher Scientific
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Gene synthesis is a laboratory equipment used for the creation and production of synthetic DNA sequences. It enables the design, construction, and verification of custom DNA fragments, which can be used for various applications in molecular biology, biotechnology, and genetic engineering.
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Lab products found in correlation
Peg it solution, by System Biosciences (3 mentions)
Pcdna3, by Thermo Fisher Scientific (2 mentions)
In fusion cloning method, by Takara Bio (1 mentions)
Quikchange method, by Agilent Technologies (1 mentions)
Kod polymerase, by Merck Group (1 mentions)
Pmd2 g envelope plasmid, by Addgene (1 mentions)
Pultra, by Addgene (1 mentions)
Fugene hd transfection reagent, by Addgene (1 mentions)
Pspax2 packaging plasmid, by Addgene (1 mentions)
Methotrexate, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Cho s cells, by Thermo Fisher Scientific (1 mentions)
Cedex bio analyzer, by Roche (1 mentions)
Pcdna3 vector, by Thermo Fisher Scientific (1 mentions)
Pcdna3 flag mettl3, by Addgene (1 mentions)
Z vad fmk, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Mg132, by Cayman Chemical (1 mentions)
Dna sequencing, by Eurofins (1 mentions)
11 protocols using gene synthesis
1
Generation and Characterization of Dll1 and Dll4 Constructs
2
Generation and Cloning of Bacterial Deubiquitinases
3
Lentiviral production of GC-C variants
4
Preparation of Trastuzumab-Based Antibodies
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The trastuzumab- and trastuzumab-IFN-β mutein-expressing gene constructs were generated by gene synthesis (Cosmogenetech, Seoul, Korea), and the synthesized heavy and light chain DNAs were inserted into the pCHO 1.0 expression vector (Life Technologies, Gaithersburg, MD, United States) at the AvrII-Bstz17I and EcoRV-PacI sites of the polylinker region, respectively (Supplementary Table S1 ). CHO-S cells (Life Technologies, Gaithersburg, MD, United States) were transfected with the expression vectors, and stable clones were selected with 100–10,000 nM of methotrexate (Sigma, NY, United States) and 10–50 μg/ml of puromycin (Life Technologies, Gaithersburg, MD, United States). Culture media from CHO-S cells stably expressing trastuzumab and trastuzumab-IFN-β muteins were collected and loaded onto a MabSelect SuReTM rProtein A agarose-bead resin (GE Healthcare, WI, United States), and the proteins were eluted using 0.1 M sodium citrate buffer (pH 3.0). The purified antibody and antibody-cytokine fusion proteins were quantified using the Cedex Bio Analyzer (Roche, Indianapolis, IN, United States) and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions.
5
Cloning of hCMV iE1 Proteins
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The cDNAs encoding hCMV iE11–328 and iE11–289 fused to an N-terminal FLAG tag were obtained by gene synthesis (Integrated DNA Technologies, Inc.) and then cloned into the pCDNA.3 vector (Invitrogen) (pCDNA3-FLAG-hCMV iE11–382 [BUG 3779; Bacteria-Cell Interactions laboratory's bacteria collection] and pCDNA3-FLAG-hCMV iE11–289 [BUG 3780]). The plasmids encoding YFP-CBD, a yellow fluorescent protein (YFP) chimera protein of the cell wall binding domain (CBD) from the Listeria phage endolysin Ply118 (BUG 2305; kind gift from J. Swanson, University of Michigan Medical School, Ann Arbor, MI, USA), and Sp100 (pSG5-Sp100WT [BUG 4134] and its derivative pSG5-Sp100K297R [BUG 4135]) have been described previously (11 (link), 57 (link)).
6
Plasmid Constructs for FMDV Protease and LGP2 Studies
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Plasmids encoding the WT or C51A mutant Lb protease were generated by PCR amplification of the corresponding regions from an FMDV O1K full-length cDNA clone [46 (link)] and insertion into the BamHI and XbaI sites of pcDNA3.1(+) (Invitrogen). Plasmids encoding the sequence of porcine LGP2 with a C-terminal Myc tag and/or an N-terminal DDK tag were generated by gene synthesis (NZYTech) and cloning into the NheI and XbaI sites of pcDNA3.1(+) (Invitrogen). Plasmid encoding (C-terminal Myc-DDK-tagged)-human LGP2 was from Origen. Plasmid pcDNA3/Flag-METTL3 encoding human methyltransferase-like 3 was from Addgene (# 53739).
For transfection, 2 μg of LGP2-encoding plasmids and 1 μg of plasmids encoding FMDV proteases were used using Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendations. The total amount of transfected DNA was balanced to 3 μg with empty vector. In some experiments, the transfection medium was supplemented with 20 μM Puromycin (apoptosis inducer, Sigma-Aldrich), 20 μM zVAD-FMK (broad caspase inhibitor, Promega) or 10 μM MG132 (proteasome inhibitor, Cayman Chemical).
For transfection, 2 μg of LGP2-encoding plasmids and 1 μg of plasmids encoding FMDV proteases were used using Lipofectamine 2000 (Invitrogen) following the manufacturer's recommendations. The total amount of transfected DNA was balanced to 3 μg with empty vector. In some experiments, the transfection medium was supplemented with 20 μM Puromycin (apoptosis inducer, Sigma-Aldrich), 20 μM zVAD-FMK (broad caspase inhibitor, Promega) or 10 μM MG132 (proteasome inhibitor, Cayman Chemical).
7
Generation of Expression Plasmids for Pou5 Rescue Experiment
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Expression plasmids carrying Pou5 coding sequences (CDS) were generated for ZHBTc4 ESC rescue experiment by inserting the triple flag-tagged (3xflag) Pou5 coding sequences into pCAGIP vector43 (link),69 (link) between the CAG promoter and the IRES-PAC (Puromycin resistant gene encoding puromycin N-acetyl-transferase). The sources of Pou5 genes used for the rescue assay are listed in Supplementary Data 2 . Pou5 CDS for CpP1, CpP3, EbP5, LcP1, LcP3, LeP1, LeP3, MeP1, MeP3, RtP1, RtP3, ScP3 and chimeric constructs S313, EbP5LH2 and EbP5S4LH2 were synthesised by gBlock (IDT) and Gene synthesis (Invitrogen) services. XhoI/NotI sites were used to insert Pou5 fragments into the pCAG 3xflag mOct4 vector in replace of the mouse Oct4 CDS. For LcP1, AmP1, AmP3 with XhoI sites present in the CDS, GeneArt® Seamless Cloning & Assembly (Invitrogen) was used to subclone the Pou5 CDS into pUCL19 carrying a 3xflag sequence. The 3xflag Pou5 CDS were then inserted by transfer a XbaI/NotI fragment into the same sites in the pCAG vector. DNA sequencing was performed by GATC Biotech.
8
Generation and Purification of Fab-fragments
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Monomeric Fab-fragments originating from monoclonal antibodies were generated by gene synthesis (Invitrogen, Carlsbad, California) or by PCR-based cloning of the variable region from hybridomas as previously described [16] . In brief, the obtained variable sequences were fused with constant domains of human subclass IgG1/k with the heavy chains carboxy-terminally fused with a Streptag affinity tag and verified by sequencing. All combinatorial cloning was done using the StarGate cloning system (IBA, Göttingen, Germany) with fusion vectors adapted for Fab expression. For affinity modulation of the generated Fab-fragments mutagenesis PCR was applied to introduce targeted amino acid substitutions. Following cloning, the Streptag fusion proteins were periplasmatically expressed in E. coli K-12 strain JM83 and subsequently purified from periplasmic extracts by Strep-tag/Strep-Tactin affinity chromatography via a StrepTactin Superflow column (IBA) and stored in PBS pH 7.5.
9
Recombinant CCN Fusion Protein Expression
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Ubiquitous chromatin opening elements-CMV plasmid expression vectors encoding fusion protein with full-length CCN5 (HHS-CCN5(FL)), fusion proteins with the TSP1 homology domain of CCN5 (HHS-CCN5(III) or Alb-CCN5(III)) or CCN3 (Alb-CCN3(III)), or the HHS fusion partner (HHS) alone were generated by recombination cloning using the Gateway cloning system. The expression vectors also encoded dihydrofolate reductase allowing selection pressure with methotrexate. Maps of the expression vectors with inserts are shown in Fig. S1 , A and B. Inserts were codon-optimized for expression in hamster cells and synthesized as “DNA strings” or “gene synthesis” (Thermo Fisher Scientific). DNA string constructs were verified by DNA sequencing (Eurofins Genomics), and DNA sequences of constructs generated by gene synthesis were verified by the manufacturer.
10
Codon-Optimized BHAC Gene Expression
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BHAC genes were codon optimized for expression in A. thaliana by gene synthesis (ThermoFisher Scientific) and matured for golden-gate cloning. All plasmids were generated with the MoClo tool kit, including vector backbones and genetic parts (51 (link)). Plasmids were sequenced by Sanger sequencing (Microsynth). Plasmids and primers used in this study are listed in SI Appendix, Tables S1 and S2 , respectively.
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