So our model allows study of the post-transcriptional modulation of a specific transcript in alveolar epithelial cells in primary culture and their physiological and pathophysiological conditions. So the transient transfection of primary alveolar epithelial cells has minimal effect on cell physiology and metabolisms, which poses a clear advantage over classic protocols using transcription inhibitors. For the generation of a response plasmid expressing a gene of interest, the sequence of the gene, and the multiple cloning sites of the inducible vector have to be analyzed to identify the recognition sequences in the multiple cloning sites that are not present internally in the gene.
Using high fidelity taq polymerase and standard overlap PCR techniques, flank the gene of interest with two selected restriction enzyme recognition sites using designer primers. A sequence in coding the V5 epitope upstream of the gene of interest must be included to distinguish the expression of the transfected gene from endogenous expression. Mutants can be generated by sequential deletion to study the effects of different three prime untranslated regions on the stability of the mRNA of the gene using reverse primers encoding a polyadenylation site that gradually deletes the three prime end of the untranslated region.
Ultimately, the insertion of the gene of interest can be confirmed by restriction analysis. The gene orientation and the absence of mutations potentially introduced during rtPCR can be confirmed by sequencing. For the transfection of primary rat lung type two alveolar epithelial cells, seed the cells at a one times 10 to the sixth cells per square centimeter in 100 millimeter Petri dishes in the presence of complete minimum essential medium.
Culture for 24 hours at 37 degrees Celsius in 5%carbon dioxide with humidity. The next day, add 500 microliters of complete medium without antibiotic to each well of a new 12 well plate and pre-warm the plate at 37 degrees Celsius for 30 minutes. During this incubation, add one microgram of the response plasmid and one microgram of regulatory vector to one 1.5 milliliter tube per well.
Wash the alveolar epithelial cells with 10 milliliters per well of 37 degrees Celsius PBS without calcium or magnesium and treat the cells with five milliliters per well of 37 degrees Celsius 0.05%trypsin for two to four minutes in the cell culture incubator. When the cells have detached, neutralize the trypsin with 10 milliliters per well of complete medium without antibiotic and collect the cells into a new 50 milliliter tube. Wash the dish with four milliliters of fresh medium to collect any remaining cells and sediment the cells by centrifugation.
Resuspend the pellet in one milliliter of PBS for counting and centrifuge the cells again. Resuspend the pellet at a density of four times 10 to the seventh cells per milliliter of resuspension buffer and add four times 10 to the fifth cells to each tube of plasmid and vector with gentle mixing. Place one tube in the electroporation device and fill the tube with 3.5 milliliters of electrolytic buffer.
Fully depress the piston to insert a gold plated electrode tip into a pipette and gently mix the tube contents. Carefully aspirate the cells with a pipette and insert the pipette into the electroporation station until a clicking sound is heard. Select the appropriate electroporation protocol for alveolar epithelial cells and press Start on the touchscreen.
Immediately after the transfection, remove the pipette and transfer the cells into one well of the pre-warmed 12 well plate. When all of the cells have been electroporated and plated, place the plate in the cell culture incubator, replacing the supernatant in each well with complete medium with antibiotics after two days. The success of the transfection can be confirmed by the expression of EGFP as observed by fluorescence microscopy or flow cytometry using a control vector.
To inhibit the transcription of the gene of interest, 72 hours post-transfection replace the supernatant with one milliliter per well of complete medium supplemented with one microgram per milliliter of freshly prepared adoxycycline. To assess the mRNA half-life of the gene of interest, return the plate to the cell culture incubator from 15 minutes to six hours. Washing one well with one milliliter of ice cold PBS before lysing the cells with 500 microliters of lysis buffer from a commercially available phenol chloroform RNA extraction kit and gentle shaking at each experimental time point.
Then, isolate the RNA according to kit instructions and determine the RNA yield and purity by spectrophotometry at 230, 260, and 280 nanometers. To determine the stability of the isolated RNA, first treat one microgram of the total RNA from each sample with RNase free DNase one to remove any plasmid DNA traces. Use a commercially available cDNA synthesis kit with a blend of oligo dT and random hexomer primers to improve the reverse transcription efficiency to reverse transcribe the DNA depleted total RNA into cDNA according to manufacturer's instructions.
Add 180 microliters of molecular biology grade water to the 20 microliters of the reaction mix to dilute the cDNA reaction at a five nanogram per microliter concentration and immediately dilute the cDNA mix with fresh molecular biology grade water to reach a 1.25 nanogram per microliter concentration. Combine five microliters of cyber green dye master mix with 0.1 microliter of molecular biology grade water, 0.45 microliters of 7.5 micromolar forward primer, 0.45 microliters of 7.5 micromolar reverse primer, and four microliters of 1.25 nanogram per microliter of cDNA to obtain a total reaction volume of 10 microliters. Place the samples in a qPCR thermocycler and amplify the V5 tag gene of interest and tetrocycline transactivator advance amplicons using the qPCR conditions as indicated and generate a high resolution melting curve to assess the specific melting temperatures of the desired amplicons and to ensure the absence of noise amplicon peaks.
This pipette electroporation technique facilitates a 25 to 30%transfection efficiency rate as observed by the ratios of EGFP cells detected by fluorescence microscopy and flow cytometry. The treatment of alveolar epithelial cells with one microgram per milliliter of doxycycline has no significant impact on the expression of endogenous alpha ENaC mRNA at any time point during the treatment period. Using a transcriptionally controlled plasmid expression system as demonstrated, V5 alpha ENaC signal could be normalized according to the tetracycline transactivator advanced signal to determine the efficiency of transfection using the double delta quantification cycle method.
The half-life of alpha ENaC mRNA is up to seven times shorter in the presence of the tetracycline off system than in the presence of actinomycin D, confirming that actinomycin D leads to an artifactual alpha ENaC mRNA stabilization. Further, cyclohexamide and tumor necrosis factor alpha treatment significantly decreased the stability of the transcript while lipopolysaccharides do not. Significant changes in the modulation of V5 alpha ENaC mRNA stability can be induced depending on the deleted and included regions of the three prime untranslated region.
In addition, the over expression of specific RNA binding proteins decreases the stability of 5-alpha ENaC mRNA compared to transfection with an empty pcDNA three plasmid. This tool will be useful for querying novel insights into the post-transcriptional regulation of key genes involved into the function of alveolar epithelium and their physiological or pathological conditions.
