title: Protein transfection of mouse lung.
abstract: Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes(1). In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice(2,3) or viral or non-viral vectors that elevate protein levels via increased gene expression(4). Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model(5). While conditional transgenics avert problems associated with chronic gene expression(6), the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement(7). As with transgenics, the use of viral and non-viral vectors is expensive(8) and can provoke dose-dependent inflammatory responses that confound results(9) and hinder expression(10). Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector(11,12). Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung(13). Using β-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 μl of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice(14). The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity(15). Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27 ± 4 control vs. 31 ± 5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.
authors.affiliation: Department of Medicine, St. Luke's Roosevelt Medical Center.
authors.forename: Patrick
authors.initials: P
authors.lastname: Geraghty
authors.forename: Robert
authors.initials: R
authors.lastname: Foronjy
mesh_terms: D000818:Animals; D018893:Bronchoalveolar Lavage / Q000379:methods; D008055:Lipids / Q000008:administration & dosage*; D008081:Liposomes / Q000008:administration & dosage; D008168:Lung / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D014162:Transfection / Q000295:instrumentation / Q000379:methods*; D001616:beta-Galactosidase / Q000008:administration & dosage*
chemical_list: D008055:Lipids; D008081:Liposomes; D001616:beta-Galactosidase