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M. Wang, D. C. Otteson, L. J. Frishman; Intracellular Delivery of Proteins into Müller Glia Cells in vitro and in vivo Using Chariot Transfection Reagent. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1687. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the capability and efficiency of Chariot to deliver proteins and antibodies into Müller cells in vitro and in vitro.
Mouse Müller cells were grown to 80% confluence in 24-well dishes in Neurobasal, B27, 20mM glutamine, 2% FBS. Chariot/protein complexes containing ß-galactosidase (ß-gal) enzyme (0.5-1µg/well) or rabbit IgG antibodies (5.75- 46µg/well) were prepared and added to cells according to manufacturer’s instructions. After 1-3 hours incubation, cells were fixed and processed for ß-gal histochemistry or immunostained with Alexa488 anti-rabbit secondary antibodies. Chariot or Chariot/IgG complexes were injected in vivo into the vitreous of adult mice (1µl per eye) and dark-adapted ERGs were recorded. Cyosections were processed for immunohistochemistry using mouse anti-glutamine synthetase (GS) primary, followed by Cy3- and Alexafluor-conjugated secondary antibodies respectively. Digital images were captured using an Olympus XI71 inverted or Leica confocal microscope.
Chariot successfully delivered active ß-gal enzyme into cultured Müller cells with high efficiency, with 80 % (0.5µg ß-gal/well) to 90 % positive cells (1µg ß-gal/well). Three hours incubation yielded the highest efficiency with no detectible cytotoxicity, although the intensity of ß-gal staining varied among cells. Uptake of non-specific IgG antibodies by Müller cells in vitro increased with antibody concentration. Immunostaining showed relatively even distribution of IgG within the cells. Following intraocular injection of Chariot/IgG complexes, retinal histology was well preserved and IgG was detected across the entire length of cryosections and in all retinal lamina. Staining was most intense near the vitreous surface. Co-localization of IgG and GS immunoreactivity identified some positive cells as Müller glia. Following intraocular injection of Chariot alone or Chariot/IgG complexes, the maximum amplitude of the b-wave and scotopic threshold response of the ERG was reduced by about 40% in both injection conditions compared to control eyes.
Chariot efficiently delivers active ß-gal enzyme and rabbit antibodies into cultured Müller cells in vitro. Intravitreal injection of Chariot/IgG complexes can also deliver antibodies into retinal cells including Müller glia in vivo. The technique has potential utility for intracellular delivery of antibodies to membrane proteins and other cellular components.
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