Human furin was cloned in the sleeping beauty transposon plasmid (30 (link)) pSB-bi-RP (Addgene, catalog no. 60513), transfected along with transposase and pCMV(CAT)T7-SB100 (Addgene, catalog no. 34879) into Vero cell using PEI Max (MW 40,000; Polysciences), and selected with 2.5 μg/mL Puromycin (Gibco). Clonal cell lines were generated through limited dilution of the polyclonal cell line on a 96-well plate at the concentration of 0.3 cell/well.
Psbbi rp
Manufactured by Addgene
Sourced in United States
The PSBbi-RP is a lab equipment product. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Pcmv cat t7 sb100, by Addgene (3 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Pei max, by Polysciences (1 mentions)
Pbabe neo htert, by Addgene (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Psbbi gn, by Addgene (1 mentions)
Pbabe neo largetcdna, by Addgene (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Psbbi rp cov2 rbd puro, by Addgene (1 mentions)
4 protocols using psbbi rp
1
Cloning Human Furin in Sleeping Beauty
2
Immortalization of Cells Using Plasmids
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Plasmids (pCMV(CAT)T7-SB100 (#34879), pSBbi-GN (#60517), pSBbi-RP (#60513), pBABE-neo-hTERT (#1774), pBABE-neo largeTcDNA (#1780) were purchased from Addgene. cDNAs were PCR amplified and cloned into the pSBbI vectors via either directional SfiI cloning for hTERT [12 (link)] or by cloning using the In-Fusion® HD Cloning Kit (Clontech, Mountain View, CA, USA) for SV40 large T antigen (SV40LT). Primer sequences are listed in Additional file 2 : Table S2. Immortalization vectors contained either hTERT or SV40LT under the control of the EF1α promotor as well as a GFP/RFP-2A-puromycin/neomycin selection cassette under the control of the synthetic RPBSA promoter (Additional file 1 : Figure S1).
3
Salmonella-based Oral Vaccine Vector Development
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Salmonella typhimurium MvP728 (ΔhtrA/ΔpurD) has function as an oral vaccine vector (10 (link), 23 (link), 45 ). Bacteria were cultured in Luria-Bertani (LB) with or without chloramphenicol and/or carbenicillin (50 μg/ml).
The pSBbi-RP (#60513) (42 (link)) and pCMV(CAT)T7-SB100 (#34879) (46 (link)) plasmids for the Sleeping Beauty transposon system were from Addgene and kindly donated from the Kowarz and Izsvak Laboratories. The two plasmids were transformed into competent double mutant S. typhimurium MvP728 using electroporation (Bio Rad) after which sensitivity to carbenicillin and chloramphenicol was employed for selection.
Mouse tgfb1 sequence was amplified from pCMV6-Entry-tgfb1 (Origene, MR227339) using forward primer 5′-CCATGGATGCCGCCCTCGGG-3′ and 5′-AAGCTTTTAAACCTTATCGTCGTCATCCTTG-3′ reverse primer. The PCR fragment was cloned using TOPO TA cloning kit (Thermo fisher, 450641) and confirmed by sequencing. Next, the tgfb1 sequence was cut by NcoI/HindIII then subcloned into NcoI/HindIII digested pSBbi-RP plasmid to yield pSBbi-RP-TGFβ (Figure 1A ). The pSBbi-RP-TGFβ and pCMV(CAT)T7-SB100 plasmids were transformed into Salmonella as described (47 (link)).
The pSBbi-RP (#60513) (42 (link)) and pCMV(CAT)T7-SB100 (#34879) (46 (link)) plasmids for the Sleeping Beauty transposon system were from Addgene and kindly donated from the Kowarz and Izsvak Laboratories. The two plasmids were transformed into competent double mutant S. typhimurium MvP728 using electroporation (Bio Rad) after which sensitivity to carbenicillin and chloramphenicol was employed for selection.
Mouse tgfb1 sequence was amplified from pCMV6-Entry-tgfb1 (Origene, MR227339) using forward primer 5′-CCATGGATGCCGCCCTCGGG-3′ and 5′-AAGCTTTTAAACCTTATCGTCGTCATCCTTG-3′ reverse primer. The PCR fragment was cloned using TOPO TA cloning kit (Thermo fisher, 450641) and confirmed by sequencing. Next, the tgfb1 sequence was cut by NcoI/HindIII then subcloned into NcoI/HindIII digested pSBbi-RP plasmid to yield pSBbi-RP-TGFβ (
4
SARS-CoV-2 RBD Expression Construct
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The coding sequence of the SARS-CoV-2 RBD (Wuhan-Hu-1 isolate, GenBank: QHD43416.1, amino acids 319–541) with the spike signal peptide at the N-terminus and a C-terminal histidine tag (Amanat et al., 2020), was human codon optimized, commercially synthetized and cloned (GenScript, Piscataway, NJ, USA) into the NcoI and XbaI sites of the transposon-based vector pSBbi-RP (a gift from Eric Kowarz, Addgene plasmid #60,513) [32 (link)] to generate the plasmid pSBbi-RP-CoV2/RBD-Puro (Addgene plasmid #161,793). To increase the efficiency of translational initiation, a glycine was added at the second position of the signal peptide (MG
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