Online Depository
Chronological expression of Ciliated Bronchial
Epithelium 1 during pulmonary development
Hans Michael Haitchi1, Hajime Yoshisue1, Anna Ribbene1,3, Susan J.
Wilson1, John W. Holloway1,2, Fabio Bucchieri1,3, Neil A. Hanley2, David
I. Wilson2, Giovanni Zummo3, Stephen T. Holgate1, Donna E. Davies1
1
Infection, Inflammation and Repair and 2Human Genetics Divisions, Southampton
University School of Medicine, Southampton General Hospital, Southampton, United
Kingdom, 3Human Anatomy Section, University of Palermo, Palermo, Italy
*Address for correspondence:
Professor Donna E Davies
Division of Infection, Inflammation and Repair,
School of Medicine,
University of Southampton
Southampton General Hospital,
Tremona Road,
Southampton, SO16 6YD,
United Kingdom
Tel.: +44 (0)23 8079 8523
Fax: +44 (0)23 8051 1761
Email:donnad@soton.ac.uk
Running title: Cbe1/CBE1 expression during lung development.
Methods
Cloning and characterization of Cbe1 cDNA
Specific primers were designed according to the sequence information obtained in
silico (http://www.ncbi.nlm.nih.gov/Genbank/GenbankSearch.html). cDNA from
adult mouse lung was amplified by Pfu Turbo DNA polymerase (Stratagene; La Jolla,
CA) using 5’-TAGAGCTTAAGGTACCACGTGTGCC-3’ as a sense and 5’TGGTGAAGAGTTTATTGAGCCAC-3’ as antisense primers. The resulting 0.58 kb
fragment was cloned into the pPCRScript Amp vector (Stratagene).
Twelve
independent clones were subjected to dideoxy-chain termination sequencing reactions
and the reaction products were analyzed using a 377 DNA sequencer (Applied
Biosystems; Foster City, CA) by Macrogen (Seoul, South Korea).
Expression plasmids, transfection, and Western blotting
The cDNA fragment containing open reading frame (ORF) 1 of Cbe1 (126 amino
acids) was amplified by Pfu Turbo DNA polymerase using a sense primer 5’TAAAGCTTGCCACCATGGAGTCAGTTCGAGGAATGCC-3’ (underlined is a
HindIII site and double underlined is a Kozak’s consensus sequence) and an antisense
primer 5’-TTTCTAGACTAAGGTTGGGATACGGGTAAAATG-3’ (underlined is an
XbaI site), and then ligated into the HindIII and XbaI sites of pcDNA3.1 (Invitrogen,
Carlsbad, CA). An expression plasmid for ORF2 of Cbe1 cDNA (162 amino acids)
was
similarly
generated
except
that
the
antisense
primer
5’-
TTTCTAGACTAGGAAGGGTAGACAGGACAGGTC-3’ (underlined is an XbaI
site) was used.
After sequence verification of PCR-generated portions, the
recombinant plasmids were purified by the EndoFree preparation system (Qiagen;
Crawley, UK). Human embryonic kidney (HEK) 293 cells were seeded at 1 x 106
cells/well in 6-well dishes and 2 g/well of the expression plasmid was transfected
using Lipofectamine 2000 (Invitrogen). Isolation of cellular extracts, SDS-PAGE and
Western blotting were performed as described previously (1).
Semi-quantitative RT-PCR
All RNA samples were isolated using Trizol reagent according to the manufacturer’s
instructions and treated with RNase-free DNase I (Ambion; Huntingdon, UK) to
remove possible contamination with genomic DNA.
cDNA was synthesized as
previously described (1). For semi-quantitative PCR, cDNA was amplified using
specific primers; primers for CBE1, FOXJ1 and TEKT1 were as previously described
(1). For nested PCR, the following internal primers were used: CBE1 sense:- 5’CGAGGAATGCCCTTGGAATG-3’ and
antisense: 5’-CGAGCAAGCACTTTCGTAACCATG-3’; and TEKT1 sense: 5’AGCTGGCTGATCATCTGGCCAAG-3’
and
5’-
antisense
TTCGTCGATATAAATGGTGTTC-3’. For the detection of Cbe1 the following
primers were used, 5’-AGAGCTTAAGGTACCACGTGTGCC-3’ (sense, Cbe1-S)
and 5’-TTGTAACAGCACATTGCATTCCTG-3’ (antisense, Cbe1-AS). In order to
specifically detect the ORF1 and ORF2 isoforms of Cbe1 mRNA, the sense primer
Cbe1-S
and
the
following
antisense
primers
were
used;
5’-
ACCGAAGGGCGACACACTCAGTCG-3’ (Cbe1-ORF1-AS) for ORF1 and 5’ACCGAAGGGCGACACACTCCTTAC-3’ (Cbe1-ORF2-AS) for ORF2. For the
detection of the longer forms of Cbe1 mRNA, the sense primer 5’AGCATCCCTGAGACCCTACCATG-3’ and the antisense primer Cbe1-AS, Cbe1ORF1-AS, or Cbe1-ORF2-AS were used. For an internal standard, expression of
glyceraldehyde-3-phosphate dehydrogenase (G3pdh) was also analysed.
PCR
products were separated in 1.5% agarose gel and visualised with ethidium bromide or
Vistra Green (Amersham Biosciences; Amersham, UK).
Reverse transcription quantitative real-time PCR (RT-qPCR)
RT-qPCR was performed using an IcyclerIQ system (Bio-Rad; Hemel Hempstead,
UK) (2). Relative expression levels were calculated using the CT method. At least
five independent tissue samples were analysed per condition, and each cDNA sample
was measured in duplicate. Specific primers and probes are provided in Table 1.
Each reaction consisted of 6.5 L of reaction mixture (Eurogentech; Seraing,
Belgium), 1 L of a primer mixture (each 7.5 M), 0.375 L of SYBR green
(Eurogentech), 0.125 L of 1 M fluorescein (Eurogentech), 2 L of sterile water, and
2.5 L of cDNA solution (total 12.5 L). The results were expressed relative to either
Gapdh or ACTB mRNA levels which were used as house keeping genes.
Table 1: Primers sequences for qPCR assays
Target gene
CBE1
TEKT1
FOXJ1
Cbe1*
Foxj1*
Foxa1*
Foxa2*
Tekt1*
* SYBR green assays
Primer/probe sequences
5’-TCCATGGTTACGAAAGTGCTTG-3’ (sense)
5’-CCGCTGGCGTAGTAGTCCA-3’ (antisense)
5’-TCGGGCCGCCACTACTGTCTGCT-3’ (probe)
5’-AAACCTCAAATGGCTAAACTATTACAAC-3’ (sense)
5’-CCTCTGGCTTTCTGCGACC-3’ (antisense)
5’-TCCACCCAAGTTCCTGCCCTCAGAGT-3’ (probe)
5’-GCCCAGGACCAGAATCGCT-3’ (sense)
5’-GGAAGACGCGGAGCAATGAAACAC-3’ (antisense)
5’-CCTCCTCTCCCCAGCCCCACCTTGT-3’ (probe)
5’-TGCCACTTCCAGGACACC-3’ (sense)
5’-TCCGAGACAGCGAGTTCAG-3’ (antisense)
5’-AGAGAGTGAGGGCAAGAGAC-3’ (sense)
5’-GCGGGCTTAGAGACCATTTC-3’ (antisense)
5’-CCAGACCCGTGCTAAATACTTC-3’ (sense)
5’-TGTGGTTGGTTTGGTGTGTG-3’ (antisense)
5’-GCAGAGCCCCAACAAGATG-3’ (sense)
5’-TGAGAAAGCAGTCGTTGAAGG-3’ (antisense)
5’-GACGGATGACCTGCTTACG-3’ (sense)
5’-GATGCCCACACGCTTCTC-3’ (antisense);
References
(1) Yoshisue H, Puddicombe SM, Wilson SJ, Haitchi HM, Powell RM, Wilson DI,
et al. Characterization of ciliated bronchial epithelium 1, a ciliated cellassociated gene induced during mucociliary differentiation. Am J Respir Cell
Mol Biol 2004 Jul 8;31((5)):491-500.
(2) Powell RM, Wicks J, Holloway JW, Holgate ST, Davies DE. The splicing and
fate of ADAM33 transcripts in primary human airways fibroblasts. Am J Respir
Cell Mol Biol 2004 Jul;31(1):13-21.