Responds to Gilead Sciences’ GS 3815 which is a more stable formulation of the cytofectin GS 2888 (Figure 1) reported by Lewis, et al.3) Like GS 2888 vesicles, Cytofectin GSV can deliver plasmids as well as oligonucleotides. It should be noted that Cytofectin GSV works best with adherent cells and performs less well with cells grown in suspension culture. The following is a protocol for transfection of antisense phosphorothioate oligodeoxynucleotides using Cytofectin GSV into adherent cells grown on 100 mm plates (Figure 2). It was provided by Gilead Sciences where this system has been used for gene inhibition with C-5 propyne antisense oligonucleotides in low nanomolar concentrations.3-7 Gilead’s contribution to this project is gratefully acknowledged. Stability and Storage Conditions 1. Store Cytofectin GSV at 4; mix well before use. It is stable for at least three months. For a longer term, store at -70. Cytofectin GSV is a liquid formulation of 2 moles of the cationic lipid, Cytofectin GS, and one mole of DOPE and shipped as a 2 mg/mL lipid in sterile water. The formula weight of Cytofectin GS is
FIGURE 3: PHOTOMICROGRAPH OF CELL TRANSFECTED WITH PLASMID
Image from confocal microscopy of a cell transfected with a plasmid using Cytofectin GS and the procedures described here. We thank Tom Keller, Oregon Health Sciences University for providing us with this and other similar images.
6. Incubate 10 to 15 minutes at RT. 7. Add 3.6 mL complete growth media (10% FBS and antibiotics do not affect transfection efficiency) and mix well for a total transfection volume of 4 mL. Transfection 1. Remove media from cells and replace with the 4 mL of the oligo/lipid transfection mixture. Do not wash
cells at any time with PBS or Tris buffered saline before or after the addition of the transfection mix as this may affect transfection efficiency. 2. Incubate transfection mixture with cells for 4-6 hours, then add a further 4 mL of complete media. Do not remove the lipid/oligo transfection mixture after 4-6 hours unless intolerable toxicity is observed. 3. Dose for 24-48 hours before
PREPARATION OF CYTOFECTIN GS VESICLES1
Materials 5.284028-89-3 medchemexpress a.202138-50-9 Molecular Weight b.PMID:29262129 Cytofectin GS: (Glen 70-3815-70) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in CH3Cl (10 mg/ml): Avanti Polar Lipids, Inc., Alabaster, Alabama, # 850725* or equivalent. Store lipid preparation at 4 oC. Material retains activity for 3 months. Also material can be stored at -70 oC and will retain activity for 1 year. The liposomes can be used directly or sized by filtration through a polycarbonate membrane (50-200 nm pore sizes; Liposofast; Avestin, Ottowa, Canada). 100 nm vesicles were found to be optimal in studies by Gilead Sciences.
6.
Method 1. To one tube containing 2 mg of cytofectin GS 3815 lipid add 0.1 ml of a 10 mg/ml solution of DOPE in chloroform. Vortex to dissolve Cytofectin GS. This will yield a final molar ratio of Cytofectin GS to DOPE of 2:1. Evaporate the chloroform in a speed-vac or under an argon stream to form a thin lipid film in the tube. Add 1.5 ml sterile water to the film to form a lipid suspension. Vortex the suspension for 5 min. This yields a final lipid concentration of 2 mg/ml. Freeze-thaw the tubes 6 times (dry ice/37 oC bath).
2.
Uptake analysis of fluorescent oligonucleotides in African green monkey kidney cells (CV1), at Gilead Sciences, showed that an unsized formulation, prepared by the freeze-thaw method, of 2:1 (mole:mole) ratio of Cytofectin GS to DOPE was optimal for deliver.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
