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Nucleic Acids Research, 1991, Vol. 19, No. 12 3403-3408
© 1991

MOLECULAR BIOLOGY

p{lambda}Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning

Andrew J.M. Murphy and Robert T. Schimke

Department of Biochemistry, Imperial College of Science, Technology & Medicine Exhibition Road, London SW7 2AZ, UK

Received January 28, 1991. Revised May 10, 1991. Accepted May 10, 1991.

We have developed a new type of bacteriophage {lambda} vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call ‘selective substitution’, is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.


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