This work was supported by the National Institutes of Health (7R01HL098528-03) and FAMRI&#x2019;s Clinical Innovator Award.

1. Preparation of Protein Transfection Reagent
<ol>
	<li>Dissolve the Pro-Ject Reagent by adding 250 &mu;l of methanol or chloroform to the tube containing the dry film.</li>
	<li>Vortex for 10-20 sec at top speed.</li>
	<li>Pipette 20 &mu;l of Pro-Ject reagent into separate microcentrifuge tubes.</li>
	<li>Evaporate the solvent by placing the microcentrifuge tubes containing the Pro-Ject Reagent under a laminar flow hood for a minimum of 6 hr at room temperature. It must be completely dry. Alternatively, dry the ...

<ol>
	<li>Glasser, S. W., Korfhagen, T. R., Wert, S. E., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+models+for+study+of+pulmonary+development+and+disease.">Transgenic models for study of pulmonary development and disease.</a> Am. J. Physiol. 267, 489-497 (1994).</li><li>D'Armiento, J., Dalal, S. S., Okada, Y., Berg, R. A., Chada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+expression+in+the+lungs+of+transgenic+mice+causes+pulmonary+emphysema.">Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.</a> Cell. 71, 955-961 (1992).</li><li>Foronjy, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+expression+attenuates+cigarette+smoke-+or+elastase-generated+emphysema+in+mice.">Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.</a> Am. J. Respir Crit. Care Med. 173, 623-631 (2006).</li><li>Foronjy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+roles+of+secreted+frizzled+related+protein-1+(SFRP1)+in+lung+morphogenesis+and+emphysema.">The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.</a> Am. J. Pathol. 177, 598-607 (2010).</li><li>Costa, R. H., Kalinichenko, V. V., Lim, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+in+mouse+lung+development+and+function.">Transcription factors in mouse lung development and function.</a> Am. J. Physiol. Lung Cell Mol. Physiol. 280, 823-838 (2001).</li><li>Perl, A. K., Tichelaar, J. W., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+expression+in+the+respiratory+epithelium+of+the+mouse.">Conditional gene expression in the respiratory epithelium of the mouse.</a> Transgenic Research. 11, 21-29 (2002).</li><li>Morimoto, M., Kopan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rtTA+toxicity+limits+the+usefulness+of+the+SP-C-rtTA+transgenic+mouse.">rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse.</a> Dev. Biol. 325, 171-178 (2009).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nature reviews. Genetics. 8, 573-587 (2007).</li><li>Crystal, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Administration+of+an+adenovirus+containing+the+human+CFTR+cDNA+to+the+respiratory+tract+of+individuals+with+cystic+fibrosis.">Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis.</a> Nat. Genet. 8, 42-51 (1994).</li><li>Merkel, O. M., Zheng, M., Debus, H., Kissel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+gene+delivery+using+polymeric+nonviral+vectors.">Pulmonary gene delivery using polymeric nonviral vectors.</a> Bioconjugate Chemistry. 23, 3-20 (2012).</li><li>Yang, Y., Li, Q., Ertl, H. C., Wilson, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+humoral+immune+responses+to+viral+antigens+create+barriers+to+lung-directed+gene+therapy+with+recombinant+adenoviruses.">Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.</a> J. Virol. 69, 2004-2015 (1995).</li><li>Liu, Q., Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+inflammatory+response+to+adenovirus+vectors.">Molecular basis of the inflammatory response to adenovirus vectors.</a> Gene Ther. 10, 935-940 (2003).</li><li>Pfeifer, C., Aneja, M. K., Hasenpusch, G., Rudolph, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+9-mediated+pulmonary+transgene+expression:+effect+of+mouse+strain,+animal+gender+and+lung+inflammation.">Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation.</a> Gene Ther. 18, 1034-1042 (2011).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+regulates+innate+immune+and+proteolytic+responses+to+cigarette+smoke+exposure+in+the+lung.">Protein phosphatase 2A regulates innate immune and proteolytic responses to cigarette smoke exposure in the lung.</a> Toxicol. Sci. 126, 589-599 (2012).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Phosphatase+2a+(Pp2a)+Regulates+Innate+Immune+and+Proteolytic+Responses+to+Cigarette+Smoke+Exposure+in+the+Lung.">Protein Phosphatase 2a (Pp2a) Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung.</a> Toxicol. Sci. , (2012).</li><li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J. Appl. Physiol. 87, 2362-2365 (1999).</li></ol>

The advantage of this technique over other methods is that it produces increases in protein levels and activity within the lung tissue itself. Moreover, it penetrates to the most distal regions of the lung as opposed to staying just within the airways. We have measured increased protein activity of our injectate even after lavaging the airways with saline15. Tissue activity analyses indicate that the protein is entering cells and does not just remain in the air spaces of the mouse. This was further con...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pro-Ject</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Microsprayer</td>
 <td>Penn Century</td>
 <td>FMJ-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase antibody</td>
 <td>Santa Cruz Bio</td>
 <td>SC-19119</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse serum albumin</td>
 <td>Sigma Aldrich</td>
 <td>A3139</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine/xylazine</td>
 <td>Sigma Aldrich</td>
 <td>K113</td>
 <td></td>
 </tr>
 </tbody>
</table>

protein transfection of mouse lung

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes1. In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice2,3 or viral or non-viral vectors that elevate protein levels via increased gene expression4. 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 model5. While conditional transgenics avert problems associated with chronic gene expression6, the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement7. As with transgenics, the use of viral and non-viral vectors is expensive8 and can provoke dose-dependent inflammatory responses that confound results9 and hinder expression10. Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector11,12. Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung13.
Using &beta;-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 &mu;l 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 mice14. 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 cellularity15. Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27&plusmn;4 control vs. 31&plusmn;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.

To demonstrate the effectiveness of our technique we used a microsprayer (Penn Century) to inject the trachea of mice with 2 &mu;g of mouse albumin (Sigma) dissolved in 50 &mu;l of PBS that contained 20 &mu;l of Pro-Ject transfection reagent. The albumin treated mice were compared with mice that were treated in an identical manner with 2 &mu;g of beta-galactosidase protein (Pierce Bio). After twenty-four hours, the mice were euthanized and the lungs were processed for histological analysis. Immunohistochemistry for beta-...

Watch this Scientific Journal Video about Protein Transfection of Mouse Lung at JoVE.com

Protein Transfection of Mouse Lung

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

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JoVE Journal

Biology

9.2K Views.  St. Luke's Roosevelt Medical Center. Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.