3.3.1. Identification of CB1 Receptor Novel Exons, mRNA Isoforms and Nested RACE, RT-PCR, and Northern Blotting
1. Database searches reveal sequences of (BAC) clones that provide a genomic con-tig that includes CB1 sequences.
2. ESTs with >93% homology with the relevant 80 kb of these BAC clone sequences were also used for CB1 DNA sequence assemblies.
3.2 fold decrease of androgen regulated gene RP2 in DAT/SET-KO vs DAT-KO
6.0 fold increase of protein tyrosine phosphatase delta in DAT/SET-KO vs SET-KO
5.3 fold increase of single strand CRE-BP/Pur alpha in DAT-KO vs SET-KO
Fig. 3. Hybridization of Affymetrix Mu6500 GenChip using biotinylated cRNA probes synthesized from brain cDNAs of dopamine (DAT) and serotonin (SET) transporter gene knockout mice or double (DAT/SET) knockout mice. A gene transcript is represented by 11-16 oligomers in perfect matches (PM, upper squares) and mismatches (MM, lower squares). The hybridization intensities of the perfect matches from one knockout mouse brain are compared.
3. Two gene-specific reverse primers that corresponded to sequences in the CB1 coding region were used for nested RACE. Product bands were cloned into pCR.2.1-TOPO (Invitrogen, Carlsbad, CA) and sequenced using an ABI capillary sequencing instrument.
4. Single-strand cDNA that was reverse-transcribed from human hippocampal poly A+ RNA was used for RT-PCR reactions.
5. Partial RT-PCR products are used as Northern blotting probes. Probes are hybridized to nylon membranes that had been blotted with 40 |g of human brain total RNA or 10 |g of human hippocampal poly A+ RNA (Clontech, Palo Alto, CA).
6. After hybridization, membranes are washed twice for 20 min with 0.1X SSC/0.1% SDS at 25°C, rinsed once with water, exposed to phosphoimaging plates, stripped of hybridization probe by boiling in 0.1X SSC/0.1% SDS solution for 5 min, and rehybridized. Blots are hybridized serially with different hybridization probes.
3.3.2. Transcription Start Site and Candidate Promoter Region of CB1
1. An oligonucleotide complementary to exon 1 sequences was labeled with [y-32P]ATP (ICN Biomedicals, CA) and T4 polynucleotide kinase (Invitrogen, Carlsbad, CA) and allowed to hybridize to sequences in 5 | g human hippocampal poly A+ RNA.
2. Primer extension was performed as described by the manufacturer (Promega, Madison,WI). Radiolabeled primer extension products were separated using 6% polyacrylamide gels containing 8 M urea and co-electrophoresed size standards and detected by autoradiography.
3. Promoter activities of 5'-flanking regions were analyzed by fusing regions of different lengths to luciferase in pGL3-Enhancer (Promega, Madison, WI) to generate recombinant plasmids.
4. The promoterless negative control (pGL-blank), the positive SV-40 promoter-containing control vector pGL-SV40, and the transfection-efficiency control plasmid pRL-TK are transfected into NG108-15, N1E-115, SK-N-SH cells and CHO-K1 cells, respectively (ATCC, Manassas, VA), grown to 80% confluence in 6-well plates in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) or F12K (Sigma, Germany) by 18 h, 37°C incubation using SuperFect transfection reagent as described by the manufacturer (Qiagen, Valencia, CA).
5. Cells were incubated for another 48 h to allow expression, then collected, and lysed. Lysate luciferase activities were quantitated using the dual-luciferase reporter system (Promega, Madison, WI) and TD-20/20 (Turner Designs, Fresno, CA) as described by the manufacturer.
3.3.3 Brain-Region-Specific CB1 Isoform Expression Pattern Analysis
Human brain RNAs from different region were used as quantitative RT-PCR templates. The ratio between RT-PCR products corresponded to that of iso-forms.
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