Journal: The Journal of Neuroscience

Article Title: Neuroprotection and Neuronal Differentiation Studies Using Substantia Nigra Dopaminergic Cells Derived from Transgenic Mouse Embryos

doi: 10.1523/JNEUROSCI.19-01-00010.1999

Figure Lengend Snippet: Detection of TH expression and DA neuronal markers by Western blot analysis and RT-PCR. A, The representative DA marker, TH expression, was confirmed by Western blot analysis after maintaining the SN4741 cells for a week in high-density culture at 37°C (lane 4) and 33°C (lane 5). The 62 kDa TH band in the SN4741 cells was consistent with the molecular weight of TH isolated from mouse adrenal gland (lane 1) and similarly derived locus coeruleus noradrenergic cell line, grown at 37°C (lane 2) and 33°C (lane 3), which were used as positive controls for the Western blot analysis. Each lane contained 15 μg of protein except lane 1 (5 μg) and lane 2 (30 μg). The specific amount of TH protein in the SN4741 cells grown at 37°C was higher than the culture at 33°C. As demonstrated in a TH-positive pheochromocytoma cell, PC12, culture (Kim et al., 1995), our SN DA cell line also exhibited the increase in TH expression in the high cell density during a prolonged culture (7–10 d). B, The expression of some DA neuron-specific markers, such as NT-3, BDNF, DA-T, and D2R were detected by RT-PCR in the SN4741 cell line. After Southern blotting of both 1 and 5 μl of each RT-PCR sample, the presence of each specific size band (the strongest band) was detected and confirmed with radiolabeled human BDNF cDNA, rat NT-3 cDNA, mouse DA-T cDNA, or mouse D2R oligonucleotide probe, respectively. Each filter was hybridized with each specific probe and exposed separately. The expected major band sizes were 430 bp for NT-3, 407 bp for BDNF, 553 bp for DA-T, and 490 bp for D2R. β represents the β-actin PCR product of 289 bp as an internal standard.

Article Snippet: PCR was performed with Taq polymerase (Perkin-Elmer, Norwalk, CT) using an appropriate primer sets for BDNF (nucleotides 374–393 and 761–780; GenBank accession number G287898), neurotrophin-3 (NT-3, nucleotides 441–460 and 851–870; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"X53257","term_id":"53451"}} X53257 ), D 2 autoreceptor (D 2 R, nucleotides 1581–1600 and 2051–2070; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"X55674","term_id":"50648"}} X55674 ), and dopamine transporter (DA-T, nucleotides 2381–2400 and 6241–6250; GenBank Accession number {"type":"entrez-nucleotide","attrs":{"text":"U15791","term_id":"338819863"}} U15791 ), a β-actin internal standard for normalization (Gene Link, Thornwood, NY), trace amounts of [ 32 P]dCTP, and various amounts of cDNA.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Marker, Molecular Weight, Isolation, Derivative Assay, Southern Blot