Editing Transferin scFc formulation

Preparation of Tf–lipoplexes<br>Cationic liposomes (Lip) consisting of dioleoyl trimethyl-<br>ammonium propane (DOTAP) and dioleoyl phosphatidyl-<br>ethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL) at<br>a 1:1 molar ratio were prepared either by sonication or ethanol<br>injection as previously described (L. Xu et al., 1997, 1999a,<br>2001). Tf–Lip–DNA was prepared according to the in vivo for-<br>mulation described previously (L. Xu et al., 1999a). Briefly, for<br>a typical preparation with optimized in vivo formulation, 25 ml<br>of Tf (5 mg/ml, iron-saturated holo-transferrin; Sigma, St.<br>Louis, MO) and 50 ml of Lip (2 mM total lipids) plus 75 ml of<br>water were mixed in a polypropylene tube and incubated for<br>5–15 min at room temperature with frequent rocking. Ten mi-<br>crograms of plasmid DNA in 120 ml of 20 mM HEPES buffer,<br>pH 7.4, was added to the tube, mixed immediately and thor-<br>oughly, and incubated for 20 min at room temperature with fre-<br>quent rocking. Thirty microliters of 50% dextrose solution was<br>then added to the tube. The final DNA:lipid:Tf ratio was<br>1:10:12.5 (mg/nmol/mg). The HEPES buffer can be replaced by<br>pure water.<br>





[[Category:Transfection]]