An efficient in vivo recombination cloning procedure for modifying and combining HSV-1 cosmids.

A helper virus-free herpes simplex virus type-1 (HSV-1) plasmid vector system developed recently may have applications in gene therapy and basic physiological studies. This system might be improved by mutating specific HSV-1 genes in the packaging system and by creating large vectors. An in vivo recombination cloning procedure is reported that supports the routine manipulation of relatively large DNAs such as the five cosmids that comprise this helper virus-free HSV-1 packaging system. In vivo recombination cloning is carried out by transforming overlapping DNA fragments into a specific RecA+ Escherichia coli, BJ5183. The cloning efficiency was improved by using a modified version of the Hanahan transformation procedure, and the background was lowered by either using vectors with different combinations of ends (5' overhangs, 3' overhangs, blunt ends) or by treating the vector with calf intestinal phosphatase. The range of usable overlap sizes is from 251 bp to 18 kb with 500 bp to 5 kb preferred. This procedure supports the routine construction and mutation of HSV-1 cosmids, by use of up to six different DNA fragments, and the construction of plasmids up to 65 kb in size. This procedure may also have applications to other vector systems and to studies on large viruses.