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Adenoviral vectors are widely used to express transgenes in vitro and in vivo. A major obstacle to the generation of adenoviral vectors is the manipulation of the large (35 kb) adenoviral genome. We developed a hybrid yeast-bacteria cloning system for the creation of novel adenoviral vectors. The adenovirus 5 (Ad5) genome was cloned into a shuttle vector that contains both yeast and bacterial elements for replication and therefore functions as both a yeast artificial plasmid (YAP) and as a plasmid artificial chromosome (PAC). Any sequence can be introduced into any region of the adenoviral genome via the highly efficient homologous recombination in yeast and then these recombinants are rapidly amplified in bacteria. Adenoviral vectors are generated by introduction of the PAC into the appropriate complementing mammalian cell without the need for plaque purification. Vectors were constructed with deletions in the E1, E3, and/or E4 regions. We have generated more than 100 vectors with a number of different transgenes and regulatory elements. In addition, the YAP/PAC vector was used to capture a DNA fragment encompassing the human factor IX gene, demonstrating the utility of this system to clone and analyze genomic DNA. This novel cloning strategy allows the rapid and versatile construction of adenoviral vectors for gene expression and gene therapy applications.

With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (VH) genes, the major diversity (D) gene cluster, the joining (JH) genes, the intronic enhancer (EH), and the constant region genes, mu (C mu) and delta (C delta). Two IgK locus-derived YACs each contain three variable (V kappa) genes, the joining (J kappa) region, the intronic enhancer (E kappa), the constant gene (C kappa), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form.

A new method for screening of YAC libraries is described. Individual YACs were pooled into groups of 384 clones and prepared as samples suitable for pulsed-field gel electrophoresis. A five hit human YAC library (Brownstein et al., 1989) containing approximately 60,000 clones was condensed into 150 such pools and chromosomal DNAs in each sample were separated on three pulsed field gels containing 50 samples each. Southern blots prepared from these gels were hybridized with probes of interest to identify pools containing homologous YACs. Further purification was performed using standard colony hybridization procedures. Twenty-one probes used thus far have identified 47 positive pools and corresponding YACs have been purified from 28 of these. Some significant advantages of this method include avoidance of DNA sequence analysis and primer generation prior to YAC screening and the ability to handle the entire library on three filters. The screening approach described here permits rapid isolation of YACs corresponding to unsequenced loci and will accelerate establishment of YAC contigs for large chromosomal segments.