The changes in the P gene of the PIV5 W3 genome [T155P, S157F, K254R, N306K and S308A] were generated by primer-mediated mutagenesis using oligonucleotides purchased from Sigma and the modified fragments inserted into the rPIV5-W3 backbone plasmid, pBH276 [57 (link)] using standard molecular biology approaches. Base changes were confirmed by DNA sequencing. The pBH276-derived template plasmids (1μg) were transfected together with pCAGGS-based helper plasmids directing the synthesis of PIV5-NP (100ng), PIV5-P (100ng) and PIV5-L (500ng) into 6-well dishes containing ~106 BSRT7 cells per well using linear polyethyleneimine (PEI) of molecular weight 25,000 (Polysciences Inc., Warrington PA, USA), or Fugene, under standard conditions. Successful recovery was confirmed by immunofluorescence screening using a monoclonal antibody (PIV5-Pk) conjugated to a FITC fluorophore, which recognises PIV5 V and P [55 (link)]. Working stocks of virus were produced from positive wells by two successive passages at low multiplicity of infection (moi) in Vero cells, and stocks were harvested, clarified by centrifugation, and flash frozen in liquid nitrogen.
Linear polyethyleneimine pei
Manufactured by Polysciences
Sourced in United States, Germany
Linear polyethyleneimine (PEI) is a cationic polymer that can be used in various laboratory applications. It is a water-soluble, linear polymer that consists of repeating ethyleneimine units. PEI can be utilized as a transfection agent, a flocculating agent, or for other purposes where its cationic properties are advantageous.
Automatically generated - may contain errors
3 protocols using linear polyethyleneimine pei
1
Generation of Recombinant PIV5 Mutants
2
Fluorescent Nanogel Synthesis and Characterization
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NG used in the experimental procedures required the following polymers: polyethylene glycol (PEG; M w = 8 kDa, from Merck KGaA, Darmstadt, Germany) and linear polyethylene imine (PEI; M w = 2.5 kDa, from Polysciences Inc., Warrington, USA) used as received, without further purifications. The dyes used to produce fluorescent NG are Cyanine 5-azide (Cy5; M w 1035 Da, from Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and Rhodamine B base (RhB; M w 479 Da, by Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). RhB was modified to introduce the azide group as described in a previous study [26] (link). Solvents were of analytical-grade purity. All the rhodamine derivatives were stored at 4 • C, and Cy5 at -20 • C.
3
Formulation and Characterization of Clofazimine Delivery System
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Potassium biphthalate sodium hydroxide buffer and HPLC grade water were obtained from Fisher Scientific (Fair-lawn, NJ). Slide-A-Lyzer Mini Dialysis Units (20,000 MWCO) were obtained from Thermo Scientific (Rockford, IL). Heptakis (6-amino-6-deoxy)-β-CD heptahydrochloride (CD) was purchased from Cyclolab R&D Laboratory Ltd. (Hungary). Citric acid was from EM Science (Gibbstown, NJ). CFZ was purchased from LC laboratories (Woburn, MA). Methoxy-poly(ethylene glycol)-poly(L-glutamic acid) (PEG-PLE), methoxypoly(ethylene glycol)-poly(L-lysine) (PLL) was purchased from Alamanda Polymers (Huntsville, AL). α-Ω-bis-(amino)-terminated poly(L-histidine) (PLH) was purchased from Polymer Source Inc (Canada). Linear polyethyleneimine (PEI) was purchased from Polysciences Inc (Warrington, PA).
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