Supplemental Methods
Transfection and clonal selection:
A 3.7 kb genomic DNA fragment containing the involucrin (INV) promoter was
cloned into the pUb-Bsd vector (Invitrogen, Carlsbad, CA). The CAMP cDNA was
cloned downstream of the involucrin promoter and a DNA fragment containing the rabbit
-globin intron and poly (A) signal was inserted downstream of the CAMP cDNA. The
plasmid vectors used for modification have been designed to comply with guidance
documents on Somatic Cellular and Gene Therapy issued by CBER and are consistent
with policies specified in the NIH Guidelines for Research Involving Recombinant DNA
Molecules (2002). The structure of the INV-CAMP-Ub-Bsd construct was confirmed by
restriction enzyme mapping and DNA sequencing. To allow selection of stablytransfected NIKS® keratinocytes a gene encoding the protein that confers resistance to
blasticidin is contained on the vector backbone into which the INV promoter and CAMP
cDNA were cloned. The vector was designed to permit excision of the -lactamase
coding region prior region prior to transfection, eliminating the possibility of transmitting
resistance to clinically-relevant -lactam antibiotics such as penicillin and ampicillin.
NIKS® cells were electroporated with the CAMP construct and subsequently cultured on
a mitomycin-C treated fibroblast feeder layer in blasticidin-containing media for three
weeks. Multiple independent clones expressing the CAMP transgene were isolated,
confirmed to have transgene expression, expanded in monolayer culture for further
characterization and cryopreserved. After the initial selection, all clones were grown in
antibiotic-free keratinocyte growth medium.
Anchorage-Independent Growth Assay:
Anchorage-independent growth was determined by agar assay. NIKS®,
NIKShCAP18 or SCC4 cells were suspended at 5 x 104 cell/ml in 0.3% Noble agar as
previously described in 1. Briefly, a 1ml aliquot of each of these mixtures was pipetted
on to 1ml base layers of 0.5% Noble agar in a 9 cm2 gridded tissue culture dish. Plates
were then incubated at 37°C in a humidified 5% CO2 atmosphere. Colonies > 40µm in
diameter were counted at 3 wks after plating. NIKS® and SCC4 (a gift from Dr. James
Rheinwald) were plated in triplicate and NIKShCAP18 was plated in quintuplicate for the
experiment.
Semi-quantitative RT-PCR and Quantitative Real-time PCR Analysis:
NIKShCAP18 transgene levels in NIKS® clones surviving antibiotic selection were
measured using semi-quantitative PCR. Total cellular RNA was isolated from
subconfluent cells using Trizol Reagent according to manufacturer’s instructions
(Gibco/Invitrogen, Carlsbad, CA). Following DNase I treatment of 2µg total RNA using
the DNA free kit (Ambion, Austin, TX), reverse transcription was performed using oligo
dT primers and M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) as per
manufacturers instructions. PCR was performed using the MasterTaq Kit (Eppendorf,
Hamburg, Germany) and the following primer sets: CAMP 5’CGCCCCGGGCCACCATGAAGACCCAAAGG-3’ (forward, within human CAMP
gene) and 5’-CAACCAGCACGTTGCCCAGG-3’(reverse, within rabbit β-globin
region); GAPDH 5’-CCAGCCGAGCCACATCGCTC-3’ (forward) and 5’ATGAGCCCCAGCCTTCTCCAT-3’ (reverse). Analysis of the relative levels of CAMP
mRNA from confluent monolayer cells or organotypic tissue was measured using qPCR.
qPCR was performed using Taqman assay probes and primers for the total hCAP-18 cat
#4331182 and cyclophilin A cat #4326316E (Applied Biosystems, Foster City, CA).
Reactions were run on a PTC-thermocycler with a Chromo 4 fluorescence detector (MJ
Research now Bio-Rad, Hercules, CA). qPCR for monolayer was performed on triplicate
confluent cell monolayers. Tissue qPCR consisted of three experiments performed with
both 15 day (3 tissues) and 22 day (5 tissues) NIKS® and NIKShCAP18 tissues (total 16
tissues NIKS® and NIKShCAP18 combined). Primary keratinocyte results consisted of
three experiments performed with single tissues in each. Opticon Monitor Software and
Genex Macro (Bio-Rad, Hercules, CA) were used for data analysis. Calculated values
represent the fold expression level over NIKS® (averaged and arbitrarily set at 1) within
each experiment.
Histology and Indirect Immunofluorescence:
Human skin samples were obtained after plastic surgery under a protocol
approved by the Institutional Review Board of University of Wisconsin-Madison. Fixed
tissues or organotypic cultures were embedded in paraffin or frozen in Sakura Tissue-Tek
OCT-Compound (The Netherlands), sectioned and stained with hematoxylin and eosin by
Surgical Pathology, University of Wisconsin Hospital (Madison, WI). Alternatively, for
indirect immunofluorescence, fixed tissues or organotypic cultures were frozen in Sakura
Tissue-Tek OCT-Compound. Cryostat sections were fixed in acetone at -20°C. After
air-drying, sections were rehydrated in phosphate buffered saline. Sections were
incubated for 1 hour at 37°C with rabbit polyclonal antibodies against hCAP-18 (1:150)
(Innovagen, Sweden). Sections were then incubated for 30 minutes at room temperature
with Alexa-488 goat anti-rabbit IgG secondary antibodies at 2 µg/ml (Molecular Probes,
Eugene, OR). Finally, sections were stained for 10 minutes with 5 µg/ml Hoescht 33258
nuclear stain (Sigma, St. Louis, MO). Sections were washed with PBS, then examined
using an Olympus IX-70 inverted fluorescent microscope equipped with filters for
visualization of both Hoechst (360 nm, blue) and Alexa Fluor 488 (490 nm, green)
fluorescence using a ¼ second exposure for all images. Digital images were captured by
an Optronics DEI-750 CE camera (Goleta, CA) using ImagePro Plus software (Media
Cybernetics, Silver Spring, MD). Dual color images were created by the single color
images taken of the same field. Neonatal foreskin and adult breast skin were stained
twice with similar results. Triplicate experiments with 15 and 22 day tissues combined
displayed similar results.
Immunoblot and ELISA:
Cell lysates were obtained from tissue using CytoBuster Protein Extraction
Reagent (Novagen, Madison, WI) with or without protease inhibitors (Protease Inhibitor
Cocktail Set III, Calbiochem, San Diego, CA) as appropriate for the subsequent analysis.
Samples for immunoblot to detect hCAP-18/LL-37 contained protease inhibitors. PR-3
treated samples did not contain protease inhibitors. ELISA samples did not contain
protease inhibitors as this was found to interfere with detection. All conditioned media
samples were immediately frozen and did not contain protease inhibitors. Conditioned
media for protein analysis was collected after 24 hrs from underneath the tissue.
Immunoblots were prepared from samples and standards separated on a 4-12% NuPAGE
gel (Invitrogen, Carlsbad, CA) run under reducing, denaturing conditions. Blots were
probed with affinity-purified polyclonal rabbit anti-human hCAP-18 antibody (1:50,000)
(Innovagen, Sweden), followed by goat anti-rabbit horse-radish peroxidase (1:2000).
Blots were developed using ECL Advance Western Blotting Detection Kit per
manufacturer instructions (GE Healthcare, Piscataway, NJ). Concentrations of hCAP18/LL-37 protein present in whole cell lysate of tissues were quantitated with a
commercially available LL-37 ELISA kit (HyCult Biotechnology, The Netherlands)
using a GeniosPlus plate reader (TECAN, Research Triangle Park, NC) according to the
manufacturer’s instructions. Tissues were from two separate experiments consisting of
five tissues total.
Literature Cited:
1.
Allen-Hoffmann BL, Schlosser SJ, Ivarie CA, Sattler CA, Meisner LF, O'Connor
SL. Normal growth and differentiation in a spontaneously immortalized neardiploid human keratinocyte cell line, NIKS. J Invest Dermatol 2000; 114(3): 44455.