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

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Abstract
A series of cosmids were developed 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 30-52 kilobases. Recombinant cosmids containing the human .alpha.-globin gene cluster were isolated from these genomic libraries. The SV40 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. These cosmid vectors are potentially useful for direct isolation of structural genes.