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New cloning tools for the design of better transgenes

Gene Therapy volume 6, pages 307–308 (1999)Cite this article

Escherichia coli plays a central role in molecular biology by allowing the cloning, propagation and production of large amounts (up to hundreds of milligrams in fermentors) of highly pure foreign DNA. But cloning in bacterial host largely depends on the presence of convenient restriction endonuclease sites.1 This dependence leads to limitations on the average size that can easily be manipulated (20 kb) and on the position where the foreign DNA can be inserted. These limitations make the modification of large plasmid-based elements such as BACs and PACs2,31 very difficult.

On the other hand, homologous recombination can be achieved efficiently in the yeast Saccharomyces Cerevisiae.4 This property has led to the specific introduction of very subtle modifications (base pair change5 or addition of two base pairs6) in very large DNA molecules (up to 650 kb) cloned in yeast artificial chromosomes (YACs). However, the preparation of good quality DNA from yeast is a time-consuming process and the yields are usually very low, limiting the subsequent functional studies. An extra step is therefore required, where the modified DNA molecule is shuttled to bacteria for large scale production.7

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  1. ICRF Molecular Oncology Unit, ICSM at Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK

    Georges Vassaux

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  1. Georges Vassaux
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Vassaux, G. New cloning tools for the design of better transgenes. Gene Ther 6, 307–308 (1999). https://doi.org/10.1038/sj.gt.3300912

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