[24] Methods for molecular cloning in eukaryotic cells

Publisher Summary This chapter describes the methods for molecular cloning in eukaryotic cells. The discovery and purification of bacterial restriction enzymes have facilitated the isolation of specific deoxyribonucleic acid (DNA) fragments spanning entire operons. These DNA fragments can be joined in vitro to the replicons of Escherichia coli ( E. coli )—such as a plasmid DNA or a bacteriophage λ genome—and the replication and amplification of these molecules can be accomplished in the E. coli host cells. These methods have been used to clone a wide variety of prokaryotic and eukaryotic DNA segments in E. coli . This chapter discusses the use of papovavirus vectors for molecular cloning in mammalian cells. Several other potential cloning systems that have not yet been employed for eukaryotic cells are described in the chapter. Although the methods selected for the isolation and joining of vector and foreign DNA fragments may vary depending on the nature of their terminal sequences, one example is illustrated in the chapter to explain the way the SV40 genome is utilized for molecular cloning. The construction of hybrid molecules between SV40 DNA and the argF gene of E. coli is described in the chapter.