Abstract:
:Human cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this problems. The method relies on the use of a recently developed episomal Epstein-Barr-virus-derived cDNA expression vector (Belt et al. (1989) Gene 84, 407-417). The cloning of hypoxanthine phosphoribosyltransferase (HPRT) cDNA, corresponding to a low abundant mRNA in wild type cells is used as a model system. Size fractionated poly (A)+ RNA from wild type cells, which resulted in an approximately 10 fold enrichment in HPRT mRNA, was used to construct a cDNA library of 25,000 independent clones in the pECV25 vector. An HPRT deficient human cell line was transfected and subsequently selected with hygromycin B for DNA uptake. In a small scale experiment only 7000 hygromycin BR transfectants were sufficient to isolate 2 independent HATR clones which were shown to replicate episomes harbouring HPRT cDNA. The first insert had a 5' untranslated region (UTR) and a 3' UTR perfectly in agreement with published data. The second cDNA clone harboured an unusually long 5' UTR and a shorter 3' UTR due to alternative polyadenylation of the HPRT transcript which has not been previously recognized.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Belt PB,Jongmans W,de Wit J,Hoeijmakers JH,van de Putte P,Backendorf Cdoi
10.1093/nar/19.18.4861keywords:
subject
Has Abstractpub_date
1991-09-25 00:00:00pages
4861-6issue
18eissn
0305-1048issn
1362-4962journal_volume
19pub_type
杂志文章abstract::Transposable elements are found throughout the genomes of all organisms. Repressive marks such as DNA methylation and histone H3 lysine 9 (H3K9) methylation silence these elements and maintain genome integrity. However, how silencing mechanisms are themselves regulated to avoid the silencing of genes remains unclear. ...
journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
pub_type: 杂志文章
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更新日期:1991-12-11 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
pub_type: 杂志文章
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2017-06-20 00:00:00
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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doi:10.1093/nar/23.18.3704
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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journal_title:Nucleic acids research
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更新日期:2003-05-15 00:00:00
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pub_type: 杂志文章
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