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Published March 30, 1990 | public
Journal Article

Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning


We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the ΛEXLX(+) and λEXLX(−) vectors that can be used for the expression in Escherichia coli of protein encoded by cDNA inserts is achived by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the λSHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage λ to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage λ arms, λLOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of specialized plasmid between these items sites will convert it into a phage λ cDNA cloning vector with automatic plasmid subcloning capability.

Additional Information

© 1990 Elsevier. Received 30 July 1989, Revised 2 October 1989, Accepted 5 October 1989. We thank F.W. Studier (Brookhaven National Laboratory) for providing vectors and host strains, M. Strathmann (Caltech) and S. Elledge (Baylor College of Medicine) for providing vectors, host strains and advice, and S. Lewis for assistance with DNA sequencing. Technical assistance was provided by C. Mayeda and M. Whitney. C.H.M. thanks Martin Chaifie for providing laboratory space. M.J.P. was a postdoctoral research fellow of the Helen Hay Whitney Foundation and is a Lucille P. Markey Scholar; B.A.H. is supported in part by a USPHS Predoctoral National Research Service Award (T32 GM07616); D.D. is supported in part by a predoctoral fellowship from the California Foundation for Biochemical Research; C.H.M. is supported by a postdoctoral fellowship in Molecular Studies of Evolution from the Alfred P. Sloane Foundation and USPHS grant GM30997 to Martin Chalfie; K.V.R. was supported by a Proctor & Gamble postdoctoral fellowship and a Rockefeller Foundation Biotechnology Career Fellowship. This research was supported by a scholar's grant from the Lucille P. Markey Charitable Trust (M.J.P.), USPHS Program Project Grant GM40499 (H.D.L. and E.M.M.), Developmental Biology Grants from the Lucille P. Markey Charitable Trust (H.D.L. and E.M.M.) and the Searle Scholars Program of the Chicago Community Trust (H.D.L.).

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