PMC:64493 / 13882-16894
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/64493","sourcedb":"PMC","sourceid":"64493","source_url":"https://www.ncbi.nlm.nih.gov/pmc/64493","text":"Materials and methods\n\ncDNA clones\nHuman and mouse cDNAs were obtained from Research Genetics or the MRC Genome Resource Facility and were sequenced using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza dGTP (Amersham-Phamacia Biotech, Buckinghamshire, England) and LiCor technology (LiCor 4200 Automatic Sequencer, Lincoln, Nebraska). The human cDNA was represented by IMAGE clone 1147395; mouse cDNAs were IMAGE clones 483464, 2101532 and RIKEN #1010001J10.\n\nNorthern blot expression studies\nA PCR product derived from the 3' end of human NDNL2 was generated with oligonucleotide primers NDNL2-3F (5'-GTCTACCCCACCAAGAAGCA) and NDNL2-4R (5'-CCTTCCCCCAATCCTCTAAA), in a 20 μl PCR reaction containing 20 pmol of each oligonucleotide. The PCR was performed as follows: 94°C for 5 min. followed by 30 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 30 s, and final extension at 72°C for 10 min., The PCR product was random prime 32P-dCTP labeled with the Random Primers DNA Labeling System (Life Technologies, Rockville, MD). The labeled probe was hybridized to human adult and fetal Multiple Tissue Northern (MTN) Blots (Clontech Laboratories, Palo Alto, CA., Cat. #7760–1, 7759–1 and 7756–1) in ExpressHyb solution according to manufacturer's directions. The final wash was at 50°C in 0.1XSSC, 0.1%SDS twice for 20 min each time. Exposure to Hyperfilm (Amersham-Pharmacia Biotech) was for four days at -80°C. Oligonucleotide primers Ndnl2-1F (5'-CTTGGAGTACCGGAGGATACC) and Ndnl2-2R (5'-CAACACATCCTAACGCTCCA) were used for mouse northern blots. These primers amplified a 343 bp DNA fragment corresponding to the 3' end of the Ndnl2 gene. The PCR was performed as above but with an annealing temperature of 58°C. Mouse adult and embryo MTN blots (Cat. #7762–1 and 7763–1, Clontech Laboratories) were similarly hybridized with the radioactively labeled Ndnl21F/2R PCR product. To control for the amount of loaded RNA the same blot was subsequently hybridized with a β-actin or ubiquitin probe demonstrating approximately equal loading in all lanes.\n\nGenetic mapping and imprinting studies\nA 469 bp product corresponding to the 3' end of Ndnl2 was generated from mouse genomic DNA (C57BL/J or SPRET/Ei) using oligonucleotide primers Ndnl2-7F (5'-TGGAAACCAGCAAGATGAAA) and Ndnl2-8R (5'-AGCTACCCTGTTTCTTTATCGTC). DNA samples were sequenced on both strands using the LiCor automated sequencer. DNA amplification products were digested with MspI to produce a 469 bp undigested product in C57BL/6J and 180 bp and 289 bp digested products in DNA from M. spretus. Genetic mapping was performed on the Jackson Laboratories BSS backcross. Cell lines used for human imprinting analysis were PWS lymphoblast GM09024B and AS lymphoblast GM11515 (both from the NIGMS Human Genetic Mutant Cell Repository), PWS fibroblast 1889 from the University of Miami Brain and Tissue Bank for Developmental Disorders and AS fibroblast KAT graciously provided by Dr. A. Beaudet (Baylor College of Medicine).\n","divisions":[{"label":"Title","span":{"begin":0,"end":21}},{"label":"Section","span":{"begin":23,"end":493}},{"label":"Title","span":{"begin":23,"end":34}},{"label":"Section","span":{"begin":495,"end":2079}},{"label":"Title","span":{"begin":495,"end":527}},{"label":"Section","span":{"begin":2081,"end":3011}},{"label":"Title","span":{"begin":2081,"end":2119}}],"tracks":[]}