For transfection studies CEC (up to passage 15) were cultured in DMEM/Ham’s F12 (1:1), 5% FCS and 10 μg/ml gentamicin. Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells (Lonza) was applied according to the manufacturer’s instructions using 0.5 × 106 cells per sample. Replicate experiments were performed using cells originating from 2 different animals (aged 11 and 26 d) in order to account for inter-individual variation. For miRNA studies mimics and inhibitors were used for following miRNAs: miR-200b, miR-147b, miR-195, miR-497 (30 pmol per sample). Nonsense miRNA Pre-miR miRNA Precursor Negative Control #1 served as a negative control, Cy3 Dye-Labeled Pre-miR Negative Control #1 was used to determine transfection efficiency (all from Life Technologies).
Amaxa basic nucleofector kit for primary mammalian epithelial cells
Manufactured by Lonza
The Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells is a laboratory equipment product designed for the transfection of primary mammalian epithelial cells. It provides the necessary components to facilitate the efficient delivery of nucleic acids, such as plasmid DNA or small interfering RNA (siRNA), into these cell types.
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4 protocols using amaxa basic nucleofector kit for primary mammalian epithelial cells
1
Transfection of Bovine Epithelial Cells
2
Transactivation of Prss29 by Wt1
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PCR amplification of the Prss29 fragments was performed with the KOD polymerase kit (Novagen). The fragments were ligated into the pGL3 (Basic/Promoter) vectors (Promega). Inserts were confirmed by sequencing. Positive clones were propagated and stored at a concentration of 1 µg/µl. Primary oviductal cells were transfected using Amaxa™ Basic Nucleofector™ Kit for Primary Mammalian Epithelial Cells (Lonza). Briefly, the cell suspension in transfection reagent along with 1 µg of pGL3-Prss29 (pPrss29: 2402 bp and I4: 857 bp) reporter constructs, 1 µg of mouse Wt1 (-KTS) expression plasmid and 50 ng of pGL4.74 were co-transfected in Amaxa cuvettes using the ‘U017’ program. After transfection, the cell mixture was transferred to a 24-well plate, grown for 48 h after which the Firefly/Renilla ratio was measured using the Dual-Luciferase Assay kit (Promega) on a Mithras LB940 plate reader (Berthold). Values were normalized to either empty pGL3 reporter plasmids (for Prss29 constructs) or RcCMV expression plasmid (for Wt1 expression).
For the Wt1-R413M experiments, the R413M mutation (CGG > ATG) was introduced into murine Wt1 using the site-directed mutagenesis kit (Stratagene) and the Wt1(-KTS) encoding vector. Successful mutagenesis was confirmed by sequencing.
For the Wt1-R413M experiments, the R413M mutation (CGG > ATG) was introduced into murine Wt1 using the site-directed mutagenesis kit (Stratagene) and the Wt1(-KTS) encoding vector. Successful mutagenesis was confirmed by sequencing.
3
Reprogramming of Urothelial Cells to iPSCs
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At a cell confluence of 80–90%, UCs were harvested by trypsinization. 0.5 x 106 cells were reprogrammed using the Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells (Lonza, VPI-1005) according to the protocol of the manufacturer. 1 μg of each vector pCXLE-hOCT3/4-shp53-F, pCXLE-SK and pCXLE-hUL (addgene) was used. The cells were transferred to the Matrigel® matrix (Corning) coated well. After 24 hours, the UC medium was replaced by mTeSR-1 (Stemcell Technologies), supplemented with 1% penicilline-streptomycine and changed every second day. Cells were subcultured every 5 to 7 days. For further passaging, iPSCs were dissociated with 1 U/ml Dispase (Stemcell Technologies).
4
Transient Transfection of hTCEpi Cells
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Mammalian expression vectors encoding HA3-tagged WT K6a or serine-to-alanine mutants were introduced to hTCEpi cells using the AMAXA Basic Nucleofector kit for Primary Mammalian Epithelial Cells (Lonza). Cells at a concentration of 2 × 106 cells/100 µl of nucleofector solution containing 2 µg of plasmid were loaded into the transfection cuvette and electroporated using program U-20. Nucleofected cells were recovered in 600 µl of low Ca–KGM-2 in 2-ml Eppendorf tubes and were placed at 37°C for 30 min. Cells were plated at the desired density into 24-well plates and maintained in low Ca–KGM-2 at 37°C and 5% CO2 for 72 h before harvest.
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