The antisense ODN sequence for CPEB, 5’-CATACACCACTCCACCAAATAG-3’ (Invitrogen), was directed against a unique region of the rat mRNA sequence. The corresponding NCBI GeneBank accession number and ODN position within the mRNA-sequence are NM_001106276.1 and 1354 to 1375. The mismatch ODN sequence 5’-A ATAG AA CACA CCACCTG ATAC -3’ corresponds to the antisense sequence with 7 bases mismatched (denoted in bold).
ODN was reconstituted in nuclease-free 0.9% NaCl (10 µg/µl) and stored at −20°C until use. For each injection, rats were anesthetized with 2.5% isoflurane. A dose of 40 µg (injection volume 20 µl) of CPEB antisense or mismatch ODN were administered using a 3/10 cc insulin syringe with a 29-gauge ultra fine ½-inch fixed hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) inserted intrathecally, on the midline between the fourth and fifth lumbar vertebrae, once daily over 3 consecutive days. Using this protocol others and we have previously demonstrated the downregulation of several different proteins including the tetrodotoxin (TTX)-resistant sodium channel, NaV1.8 (Lai et al., 2002 (link)), PLC β-3 (Joseph et al., 2007 (link)), gp130, a receptor subunit for IL-6 (Summer et al., 2008 (link)) or the mitochondrial fission regulator dynamin-related protein 1 (Ferrari et al., 2011 (link)).
ODN was reconstituted in nuclease-free 0.9% NaCl (10 µg/µl) and stored at −20°C until use. For each injection, rats were anesthetized with 2.5% isoflurane. A dose of 40 µg (injection volume 20 µl) of CPEB antisense or mismatch ODN were administered using a 3/10 cc insulin syringe with a 29-gauge ultra fine ½-inch fixed hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) inserted intrathecally, on the midline between the fourth and fifth lumbar vertebrae, once daily over 3 consecutive days. Using this protocol others and we have previously demonstrated the downregulation of several different proteins including the tetrodotoxin (TTX)-resistant sodium channel, NaV1.8 (Lai et al., 2002 (link)), PLC β-3 (Joseph et al., 2007 (link)), gp130, a receptor subunit for IL-6 (Summer et al., 2008 (link)) or the mitochondrial fission regulator dynamin-related protein 1 (Ferrari et al., 2011 (link)).
