{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T01:14:04Z","timestamp":1760577244821,"version":"build-2065373602"},"reference-count":45,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2005,4,14]],"date-time":"2005-04-14T00:00:00Z","timestamp":1113436800000},"content-version":"vor","delay-in-days":3025,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Electrophoresis"],"published-print":{"date-parts":[[1997,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Progressive and selective degeneration of specific classes of neurons occurs in the Alzheimer's disease (AD) brain. Differential vulnerability in this disease is evident even within supopulations that synthesize and release acetylcholine as a transmitter; <jats:italic>i.e.<\/jats:italic>, basal forebrain cholinergic neurons degenerate but other classes of cholinergic neurons are relatively preserved. The basis for this selective vulnerability is unknown. Studies of differential neuronal vulnerability in AD would be facilitated if cell lines expressing neurotransmitter\u2010specific phenotypes could be cloned from the brain. Capillary electrophoresis (CE) with laser\u2010induced fluorescence (LIF) has been shown to be a sensitive method of detection and quantitation of the DNA products of the polymerase chain reaction (PCR). CE\/LIF was combined with the PCR to detect phenotypic messenger RNA (mRNA) molecules, converted to cDNA using reverse transcriptase (RT), in cultures of virally immortalized brainstem progenitor cells produced during establishment of a cloning strategy. RT\/PCR methods were developed for detection of the mRNAs for choline acetyltransferase (ChAT), the neuronal, constitutive isoform of nitric oxide synthase (c\u2010NOS), and the growth\u2010associated protein GAP\u201043, three genes known to be expressed in central cholinergic neurons. A \u201cnondestructive\u201d method of screening cultured cells for their expression of c\u2010NOS was established using depolarization with medium containing 50 m<jats:sc>M<\/jats:sc> potassium ion. These approaches were first validated using cultured SN56 (cholinergic) and N1E\u2010115 (c\u2010NOS\u2010positive) neuroblastoma cells, and with primary brainstem cultures. For the cloning of novel cell lines, progenitor cells were isolated from the embryonic day 13 fetal brainstem and were immortalized by transfection with a retroviral vector that confers a temperature\u2010sensitive SV\u201040 transforming activity and neomycin resistance. Cell colonies surviving in G418\u2010containing media were isolated and cloned by dilution. Clonal cultures were expanded by growth at 33\u00b0C, differentiated by switching to a low\u2010serum medium and growth at 39\u00b0C, and screened for depolarization\u2010induced accumulation of nitrite in the medium. The subset of putative c\u2010NOS\u2010positive clones (about 4%) were then screened for their expression of mRNAs using RT\/PCR in combination with CE\/LIF. This screening protocol proved to be powerful in the rapid isolation and phenotypic characterization of immortalized progenitor cells cloned from embryonic rat brainstem.<\/jats:p>","DOI":"10.1002\/elps.1150181008","type":"journal-article","created":{"date-parts":[[2005,5,28]],"date-time":"2005-05-28T11:37:48Z","timestamp":1117280268000},"page":"1750-1759","source":"Crossref","is-referenced-by-count":10,"title":["Use of capillary electrophoresis with laser\u2010induced fluorescence detection to assess messenger ribonucleic acid molecules amplified by the polymerase chain reaction: Applications in the cloning of cells"],"prefix":"10.1002","volume":"18","author":[{"given":"David","family":"Personett","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kiminobu","family":"Sugaya","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Hammond","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Robbins","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"McKinney","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2005,4,14]]},"reference":[{"key":"e_1_2_1_2_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.6338589"},{"key":"e_1_2_1_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.7901908"},{"key":"e_1_2_1_4_2","doi-asserted-by":"publisher","DOI":"10.1096\/fasebj.9.7.7737461"},{"key":"e_1_2_1_5_2","first-page":"513","volume":"147","author":"Blass J. 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