Intracellular delivery of proteins into mouse Müller glia cells in vitro and in vivo using Pep-1 transfection reagent.
Direct protein transfection is a potentially valuable tool for studying protein function in basic and clinical research. A major challenge is to enable a sufficiently large amount of protein to penetrate the plasma membrane of the transfected cells. Pep-1, a protein transfection reagent, was evaluated for its ability and efficiency in delivering proteins and antibodies into mouse Müller cells in vitro and in vivo. Pep-1 delivered active beta-galactosidase enzyme and antibodies (non-specific IgG and Cy3-conjugated anti-vimentin) into cultured Müller cells with high efficiency. Transfection efficiency increased with increasing concentration of the protein in the complex and with incubation time. Following intravitreal injection of Pep-1/IgG complexes in vivo, retinal histology was preserved and immunostaining showed that the antibodies were distributed widely across the retinal surface, with the most intense staining located near the retino-vitreal border. For complexes using non-specific IgG, double staining with anti-glutamine synthetase identified many IgG-positive cells as Müller glia. IgG immunoreactivity was also detected in the cytoplasm and occasionally in the nuclei of inner retinal neurons. Dark-adapted flash electroretinogram (ERG) recordings from injected eyes were nearly identical to ERG recordings from control eyes, suggesting that injection of Pep-1/IgG complex has minimal effects on retinal function. Therefore, Pep-1 is a useful tool for intracellular delivery of antibodies to study the role of proteins in living cells.
Wang
Minhua H
MH
College of Optometry, University of Houston, 4901 Calhoun, 505 JD Armistead Bldg Rm. 2143, Houston, TX 77204-2020, United States. mwang.2006@alumni.opt.uh.edu <mwang.2006@alumni.opt.uh.edu>
Frishman
Laura J
LJ
Otteson
Deborah C
DC
D000818:Animals; D000906:Antibodies / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D003543:Cysteamine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics; D016503:Drug Delivery Systems / Q000295:instrumentation / Q000379:methods*; D004596:Electroretinography / Q000187:drug effects; D005260:Female; D007074:Immunoglobulin G / Q000378:metabolism / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D010455:Peptides / Q000493:pharmacokinetics*; D011506:Proteins / Q000378:metabolism / Q000493:pharmacokinetics*; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D014822:Vitreous Body / Q000187:drug effects / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism / Q000493:pharmacokinetics
D000906:Antibodies; D007074:Immunoglobulin G; C503924:Pep-1 peptide; D010455:Peptides; D011506:Proteins; D003543:Cysteamine; D001616:beta-Galactosidase