Worm Breeder's Gazette 12(3): 17 (June 15, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Another l Vector for cDNA Library

Ichi Maruyama

Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA

We have constructed novel l phage vectors, lMGU, for cDNA library. The vector has the following advantages over conventional l vectors such as lgt11 and lZAP: (l) It has a positive selection property for the recombinant clones containing cDNA fragment (>104 efficiency). (2) The cDNA can be excised automatically from the l arm in vivo and can replicate as a plasmid. (3) ssDNA can be prepared from E. coli harboring the plasmid with an M13 helper phage for DNA sequencing and mutagenesis of the cDNA. (4) It can accommodate an insert up to 10 kb in size, a capacity large enough for most cDNAs.

For cDNA cloning the lMGU vector has been provided with a unique BamHI site that was created by site-directed mutagenesis of l gam gene. Insertion of cDNA into the BamHI site disrupts the gam gene activity, and the resulting recombinant l clone can grow on the P2 phage lysogen such as Q359 while parental vector phage without insert cannot. This phenotype (Spi-) has been applied for vectors such as l2001 and lEMBL for genomic library but these are all replacement vectors and, therefore. they cannot be used for small insert such as cDNA. Spi- selection allows us to select recombinant clones with more than 104-fold efficiency from packaging mixtures of recombinants and parental vectors. Hence, it is neither necessary to digest the vector DNA completely, nor to treat vectors with alkaline phosphatase to prevent self-ligation. Moreover, since non-phosphorylated synthetic oligonucleotide adaptors are used for the construction of lMGU libraries, it is virtually impossible to make recombinants that have adaptors alone as an insert or which have different cDNAs tandemly ligated. Libraries, uncontaminated by parental vectors, are also efficient for screening by plaque hybridization.

lMGU has also an automatic excision property in vivo. In lMGU genome plasmid pMGU is separated by two loxP sites from the l arms. These loxP sites are homologously recombined by Cre enzyme of bacteriophage P1 that is encoded on another plasmid in an E. coli host, IM1046 ,so that the pMGU with cDNA insert is cleaved off the arms and replicates as a plasmid. The average efficiency of recovery was about 20% and was independent of insert size. In fact, from l phage clones from a single plaque, about one hundred colonies were produced on a plate containing ampicillin. The phagemid pMGU also carries an M13 replication origin so that ssDNA can be recovered by infection with M13 helper phages such as M13K07 .This is another advantage for restriction mapping and DNA sequencing of the cDNA. Thus, using this system one can analyze and utilize recombinant clones as a l, plasmid, M13 ,depending on the purpose of the experiment.

We used the vector lMGU for the construction of a cDNA library from the mRNA prepared from a mixed population of C. elegans. From 0.1 µg of the cDNA ligated with 1.5 ug of lMGU DNAs, followed by in vitro packaging, 107 independent recombinant phages were recovered on Q359 strain. From this library, cDNAs, including mup-2 , unc-53 (T. Bogaert), unc-13 ,have been isolated. Human liver and placenta cDNA libraries have also been successfully constructed with lMGU vector (K. Hadfield).