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PMID: 6310566 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Versatile cosmid vectors for the isolation, expression, and rescue of gene sequences: studies with the human alpha-globin gene cluster.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 80 ·No. 17 ·1983-09-00 ·Pages 5225-9

Lau YF, Kan YW

Abstract

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers--SV2-gpt, SV2-DHFR, and SV2-neo--in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human alpha-globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of alpha-globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult alpha-globin genes were more actively expressed than the embryonic zeta-globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

MeSH Terms
Base Sequence Cloning, Molecular DNA Restriction Enzymes DNA, Recombinant/metabolism Escherichia coli/genetics Genes Genetic Vectors Globins/genetics Humans Nucleic Acid Hybridization Plasmids
Chemicals
DNA, Recombinant Globins DNA Restriction Enzymes
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Lau Y F
Kan Y W
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40 references, click to expand
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.80.17.5225
Published
1983-09-00
Pages
5225-9
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC384225
Subset
IM
Grants
NIADDK NIH HHS · AM16666 · United States
External Links
PubMed source DOI: 10.1073/pnas.80.17.5225 PMC Full Text View journal details
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