H-K). Open in a separate window Figure 8 LPD-mediated gene delivery to retinal ganglion cells. NeonGreen 14 (a kind gift from Dr. Martin-Paul Agbaga, OUSHC) was VP3.15 cloned into pCAGEN vector as EcoRI/Not1; this plasmid DNA was called CAG-NeonGreen. Preparation of liposome protamine/DNA lipoplexes (LPD) LPD was prepared according to the method reported previously 4, with some modification. First, the liposomes consisting of DOTAP (1, 2-dioleoyl-3-trimethylammonium-propane), DOPE (1, 2-dioleoyl-application. the transscleral route. Mice were anesthetized by intramuscular injection of a ketamine (80-100 mg/kg) and xylazine (5 mg/kg) mixture of approximately 0.1 ml, until mice did not display a blink reflex to a touch on the corneal surface. Eyes were dilated with 1% cyclopentolate hydrochloride ophthalmic solution applied to the cornea (Akron, Lake Forest, IL). The mice were kept on a 37C regulated heating pad under a surgical microscope (Carl Zeiss Surgical, NY). An insulin syringe with a beveled 30-gauge needle was used to puncture a hole in the cornea. Next, a 33-gauge blunt-end needle attached to a 10-l Nanofil? syringe controlled by a UMP3 pump controller (World Precision Instruments, Sarasota, FL) was positioned toward the superior nasal portion of the retina. Then, 1 l of LPD nanoparticles (~85 ng of DNA) were injected VP3.15 into the subretinal space. The needle was retracted 10-15 s after injection, when a bleb of retinal detachment was visible. Following VP3.15 complete removal of the injection needle, the eye was carefully observed for any indication of post-surgical complications, such as iris and sub-retinal bleeding, pronounced retinal detachment or damage, or excessive vitreous loss. After injection, saline and GelTeal lubricant eye gel (Alcon, Fort Worth, TX) were applied topically to the VP3.15 eye 3-4 times daily for 3-4 days after injection, to keep the eye continually moist. The severity of acute post-surgical complications and subsequent long-term complications, including eye infection, loss of visual function, and atrophy, were carefully evaluated to determine whether the animal would be excluded from the study. In the absence of any severe complications, the procedure was deemed successful and the animal remained in the study. Purification of TAT- fusion proteins BL21 (DE3) with the recombinant plasmid was grown to a stationary phase at 37C in LB medium containing ampicillin (100 g/ml) and a final concentration of 1 1 mM isopropyl -D-galactopyranoside (IPTG). The bacteria were harvested by centrifugation at 10,000 x g for 10 min. The bacteria were suspended in VP3.15 buffer A (50 mM Tris-HCl, pH 8.0 containing 100 g/ml lysozyme, 2 mM EDTA, 1 mM phenylmethylsufonyl fluoride, 0.5 g/ml leupeptin, 0.1% Triton X-100, 10 mM MgCl2, and 10 g/ml DNase). Rabbit polyclonal to Acinus The bacterial suspension was incubated for 30 min on ice. The lysate was cleared by centrifugation at 15,000 x g for 20 min. The pellet was discarded. The supernatant was loaded onto a Ni2+-NTA agarose (super flow) affinity column equilibrated with 10 mM imidazole. This was followed by elution with 500 mM imidazole. Transmission electron microscopy (TEM) The morphology of LPD was observed using TEM (Carl Zeiss, Germany). One drop of LPD or LPD complexed with TAT peptide was placed on a copper grid. The samples were negatively stained with 1% uranyl acetate. The grid was allowed to dry further for 20 min and was then examined with the electron microscope, as we described previously 15. Fundus imaging Mice were anesthetized with an intraperitoneal injection of ketamine (80-100 mg/kg) and xylazine (5 mg/kg) to prevent large movements during the fundoscopy (Micron III fundoscope). Both pupils were dilated using a topically applied drop of tropicamide (1%) to neutralize corneal optical power and focus the fundoscope onto the retina. Hydroxymethylcellulose ophthalmic demulcent solution (Goniosol 2.5%) was placed on.