Mechanical strain regulation of the Chicken glypican-4 gene ...

Rapid amplification of cDNA ends (RACE)
Rapid amplification of cDNA ends (RACE), to complete the 3’ region of the chicken GPC-
4, was performed with the SMART(TM) RACE cDNA amplification kit (Clontech)
according to the manufacturer’s manual. In brief, the first strand cDNA was synthesized
from chicken ESG mRNA by means of the MMLV reverse transcriptase with the aid of
SMART II oligonucleotide and 3'-RACE cDNA synthesis primers, at 42 0 C for 60 min.
PCR was then carried out with a 5’ gene-specific primer – 5’-
GCTGGAGGGGCCTTTTAACATTGAGTC-3’ – synthesized according to the cloned
969-bp fragment and the mix of two universal adapter primers provided with the kit.
Cycling conditions were: five cycles of 94 0 C for 15 s and 72 0 C for 3 min; five cycles of
94 0 C for 15 s, 70 0 C for 30 s and 72 0 C for 3 min; and 30 cycles of 94 0 C for 15 s, 68 0 C for
30 s and 72 0 C for 3 min. A nested PCR reaction was carried out under similar conditions,
with the kit primer (Nested Universal Primer) and a specific nested GPC-4 5’ primer (5'-
GAACAGCATGCAAGTGTCTCA-3'). Figure 1 describes the strategy in the assembly of
the chicken GPC-4. The obtained PCR product was separated on 1% agarose and purified,
cloned into the pGEM-T easy vector and sequenced from both directions with T7, SP6 and
specific primers. The cDNA and deduced amino acid sequences were compared with the
DNA and protein databases at the National Center for Biotechnology Information.
Preparation of riboprobe and in situ hybridization.
A fragment of chicken GPC-4 cDNA (646bp) was synthesized by reverse transcription
(RT)-PCR from the chicken ESG (see under "RT-PCR"). This fragment shared no
homology with any other known chicken cDNA and was subcloned into the pGEM-T
Easy cloning vector to produce a template for riboprobe synthesis. The sense and
antisense riboprobes were synthesized by in vitro transcription with T7 and SP6 RNA
polymerase, respectively, in the presence of linearized plasmid DNA and DIG RNA
labeling mix (Boehringer Mannheim, Ottweiler, Germany). Serial 5-µm sections of the
ESG or magnum were hybridized with a digoxigenin-labeled chicken GPC-4 probe as
described previously (25). No hybridization was observed with the sense riboprobe,
which was used as a negative control. Hybridization with avian �-actin probe was
performed in order to rule out the possibility that changes in GPC-4 expression were due
to variation in tissue processing and/or RNA preservation.
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