Name: protein transfection of mouse lung
Uploaded: 2013-05-15T12:00:00
Description: Watch this Scientific Journal Video about Protein Transfection of Mouse Lung at JoVE.com

Dette arbejde blev støttet af National Institutes of Health (7R01HL098528-03) og FAMRI s Clinical Innovator Award.

1.. Fremstilling af Protein Transfection Reagent <ol><li> Opløs Pro-Ject Reagens ved tilsætning 250 pi methanol eller chloroform til røret med den tørre film. </li><li> Vortex for 10-20 sekunder ved tophastighed. </li><li> Pipetter 20 ul Pro-Ject reagens i separate mikrocentrifugerør. </li><li> Opløsningsmidlet afdampes ved at placere mikrocentrifugerør indeholdende Pro-Ject Reagens under en laminar flow hætte i mindst 6 timer ved stuetemperatur. Det skal være helt tørt. Alternativt tørre Pro-Ject reagens v...

<ol>
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Fordelen ved denne teknik i forhold til andre metoder er, at den producerer stigninger i protein niveauer og aktiviteten inden lungevævet selv. Desuden trænger til de mest distale regioner af lungen i modsætning til opholder blot inden for luftvejene. Vi har målt øget protein aktivitet i vores injektatet selv efter lavaging luftvejene med saltvand 15.. Vævsaktivitet analyser viser, at proteinet ind celler og ikke forbliver i luftmellemrum musen. Dette blev yderligere bekræftet ved immunohistokemi viser...

<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

Stigende proteinekspression at forskerne til bedre at forstå den funktionelle rolle af dette protein i reguleringen af centrale biologiske processer 1.. I lungerne, er dette blevet opnået typisk via genetiske tilgange, der udnytter transgene mus 2,3 eller virale eller ikke-virale vektorer, der løfter proteinniveauer via forøget genekspression 4.. Transgene mus er dyre og tidskrævende at generere og den tilfældige indsættelse af et transgen eller kronisk genekspression kan ændre normal lunge udvikling og således begrænse anvendeligheden af modellen 5. Mens betingede transgene afværge problemer forbundet med kronisk genekspression 6, de omvendte tetracyclin-kontrollerede transaktivator (rtTA)-mus, som anvendes til at generere betinget udtryk, udvikle spontan luftrum udvidelsen 7. Som med transgene, er brugen af virale og ikke-virale vektorer dyre 8 og kan fremprovokere dosis-dependent inflammatoriske reaktioner, der forvirre resultater 9 og hindrer udtryk 10.. Desuden er effekten af gentagne doser begrænset af forbedrede immunreaktioner på vektoren 11,12. Forskere er ved at udvikle adeno-associerede virale (AAV) vektorer, der fremprovokerer mindre inflammation og har længere ekspression i lungen 13.. Brug β-galactosidase, præsenterer vi en metode til hurtigt og effektivt at øge protein-ekspression i lungen med en direkte protein transfektion teknik. Denne protokol blander en fast mængde renset protein med 20 ul af en lipidbaseret transfektionsreagens (Pro-Ject, Pierce Bio) for at tillade indtrængen i lungevævet selv. Den liposomale proteinblandingen injiceres derefter i lungerne af mus via luftrøret med en microsprayer (Penn Century, Philadelphia, PA). Den microsprayer genererer en fin sky af flydende aerosol hele lungerne. Ved hjælp af teknikkenvi har vist ensartet afsætning af den injicerede protein hele luftveje og alveolerne af mus 14.. Lipid transfektion teknik tillader anvendelse af en lille mængde protein for at opnå virkning. Dette begrænser den inflammatoriske respons, som ellers ville blive provokeret af højt proteinindhold administration. Faktisk bruger denne teknik, som vi offentliggjorde, at vi var i stand til at øge PP2A aktivitet i lungen uden at påvirke lungeudskylning cellularity 15.. Lungeudskylning cellularitet taget 24 timer efter udfordring var sammenlignelig med kontrol (27 ± 4 kontrol vs 31 ± 5 albumin transficeret, N = 6 per gruppe). Desuden øger protein niveauer uden at inducere lunge udviklingsmæssige ændringer eller arkitektoniske ændringer, der kan forekomme i transgene modeller. Dog kan behovet for gentagne administrationer gøre denne teknik mindre gunstig for studier af effekten af ​​langsigtede stigninger i protein udtryk. Dette ville især være True for proteiner med korte halveringstider.

<html> For at demonstrere effektiviteten af ​​vores teknik, vi brugte en microsprayer (Penn Century) for at injicere luftrøret af mus med 2 ug muse albumin (Sigma) opløst i 50 pi PBS, der indeholdt 20 ul Pro-Ject transfektionsreagens. De albumin behandlede mus blev sammenlignet med mus, der blev behandlet på samme måde med 2 ug af beta-galactosidase-protein (Pierce Bio). Efter 24 timer blev musene aflivet, og lungerne blev forarbejdet til histologisk analyse. Immunhistokemi for beta-galactosidase blev udført på...

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

Protein Transfection of Mouse Lung

Transgene mus eller virale vektorer er blevet anvendt til at øge protein-ekspression i lungen. Men disse teknikker er tidskrævende, teknisk udfordrende og har off-target effekter, der kan forvirre resultaterne. Vores protein transfektionsprotokol anvender en lipidbaserede transfektionsreagens og en ultrafin microsprayer til ensartet levere aktivt protein til lungeceller.

Protein Transfektion af Mouse Lung

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

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