INTRAMOLECULAR INTEGRATION ASSAY IN INSECT CELLS Vol 40 No. 6 November 2009 1235 Correspondence: Malcolm J Fraser Jr, Eck Insti- tute of Global Health, Department of Biological Sciences, University of Notre Dame, Indiana, 46556, USA. Tel: 574-631-6209; Fax: 574-631-7413 E-mail: fraser.1@nd.edu INTRAMOLECULAR INTEGRATION ASSAY VALIDATES INTEGRASE PHI C31 AND R4 POTENTIAL IN A VARIETY OF INSECT CELLS Jakkrawarn Chompoosri 1,2,4 , Tresa Fraser 1 , Yupha Rongsriyam 2 , Narumon Komalamisra 2 , Padet Siriyasatien 3 , Usavadee Thavara 4 , Apiwat Tawatsin 4 and Malcolm J Fraser Jr 1 1 Department of Biological Sciences, Eck Institute of Global Health, University of Notre Dame, Notre Dame, IN, USA; 2 Department of Medical Entomology, Faculty of Tropical Medicine, Mahidol University, Bangkok; 3 Department of Parasitology, Faculty of Medicine, Chulalongkorn University, Bangkok; 4 National Institute of Health, Department of Medical Sciences, Nonthaburi, Thailand Abstract. Phage φC31 and R4 integrases are site-specific and unidirectional serine recombinases. We have analyzed the ability of these integrases to mediate intramo- lecular integration between their attB and attP sites in 7 important insect cell lines as a means of predicting their relative mobility in the corresponding insect species. Both integrases exhibit significantly higher frequencies in Drosophila S2 cells than in the other insect cell lines examined, but do work well in all of the species tested. Our results, coupled with previous results of the activity of φC31 integrase in D. melanogaster and Aedes aegypti, suggest the family of serine catalyzed integrases will be useful site- specific integration tools for functional genome analysis and genetic engineering in a wide range of insect species. INTRODUCTION Practical genetic engineering of insects for potential utility as moderators of native populations requires new tools for efficient site-specific integration of genes to allow reliable prediction of expression stability and fitness costs to the transgenic insects. The most common method currently employed for engineering eukaryotic chromosomes is random integration facilitated by mobile genetic elements in which foreign or ma- nipulated DNA is introduced into the chro- mosomes of an organism without control over the ultimate position of the insertion, resulting in unpredictable gene expression and possible insertional mutagenesis lead- ing to fitness costs. In contrast, phage integration mecha- nisms can provide high specificity and have been recognized as a powerful genetic tool in a variety of prokaryotic and eukaryotic cells. While site-specific recombinases are structurally and functionally diverse (Smith and Thrope, 2002), most can be classified into either the tyrosine or serine family based on amino acid sequence homologies and cata- lytic residues. Recombinases such as Cre and FLP use a catalytic tyrosine to mediate bidirectional recombination between two identical sites (Stark et al, 1992). These recombinases rec-