Modification of the complete chicken lysozyme gene domain by poxvirus medicated gene targeting
The highly recombinant environment resulting from infection of rabbit cornea cells (SIRC) with the Shope fibroma virus (SFV) has been used to mediate the precise modification of the large chicken lysozyme gene domain. A gene targeting strategy was employed as an alternative to traditional genetic engineering practices requiring extensive sequencing and restriction endonuclease information. Homologous recombination was designed to occur between target DNA maintained in a lambda vector and modified targeting DNA maintained in a plasmid. Two target phages of differing sizes, '[lambda]DIILys' (50.7 kb, with the complete 21.5 kb chicken lysozyme gene domain subcloned into a [lambda] replacement vector) and '[lambda]ZLys4.7' (45.52 kb, with a 4.7 kb 'Spe I' fragment containing the chicken lysozyme gene start codon subcloned into a phagemid), were constructed to compare efficiencies of product recovery using the same gene targeting strategy. Three gene targeting plasmids, 'pLys.Alpha', 'pLys.GFP' and ' pLys.HT', based on modifications of the lysozyme gene start and stop codon, were designed and constructed. These constructs differed in both the extent of the modification and the amount of flanking homologous lysozyme sequence. The start codon was targeted for the insertion of 'lacZ' [alpha]-complement (584 bp, 'pLys.Alpha') and GFP (971 bp, 'pLys.GFP') coding sequences, while 'pLys.HT' was designed to insert a sequence encoding a short hydrophobic pentapeptide prior to the lysozyme stop codon. Lambda and plasmid DNA were cotransfected into SFV-infected SIRC cells; concatemerized DNA (both modified and unmodified) was recovered selectively from the cells by packaging and identified using a succession of ' E. coli' host strains. Recombination efficiencies for the insertion of the 'lacZ' [alpha]-complement sequences (from 'pLys.Alpha ') into '[lambda]DIILys' and '[lambda]ZLys4.7 ' were 5.5 and 11.9%, respectively. The 'lacZ' [alpha]-complement transferred into '[lambda]DIILys' was then "knocked out" by GFP sequences (from 'pLys.GFP') in 9.5% of the recovered phages. The transfer of the hydrophobic sequences ('pLys.HT') resulted in an apparent recombination efficiency of 3%. In comparison to ' [lambda]DIILys', SFV/SIRC gene targeting of '[lambda]ZLys4.7 ' proved to be highly efficient in both levels of recombination and eventual product recovery into plasmid form. Together, these experiments suggest that poxvirus mediated recombination constitutes a routine, rapid, and remarkably efficient genetic engineering system for the precise genetic modification of large eucaryotic gene domains when compared to traditional practices.