BAC Manipulations for Making BAC Transgene Arrays

Abstract

Chromosome tagging using lac or tet operator repeats for in vivo visualization of chromosome dynamics has now become a standard methodology used in a range of organisms. One variation of this approach has been to build transgene arrays creating artificial chromosome blocks to study various aspects of chromatin structure, transcription, replication, or DNA repair. Previously, plasmid transgenes with or without subsequent gene amplification have been used to build these arrays. However, plasmid arrays typically show heterochromatic properties, while gene amplification typically results in chromosome instability of the amplified regions. To avoid these problems, we are now building transgene arrays from large genomic DNA inserts cloned in bacterial artificial chromosomes (BAC). These BAC transgenes show transcriptional levels within several fold of endogenous genes while also exhibiting targeting to specific nuclear compartments similar to the targeting of the endogenous genes. Here we describe Tn5 transposition and BAC recombineering methods used to retrofit BACs for their use in building BAC transgene arrays. This includes insertion of operator repeats and selectable markers into these BACs as well as targeted insertion or deletion of BAC sequences.