Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond

Jonathan T Ting,Guoping Feng

doi:10.3389/fnbeh.2014.00111

Abstract

The development and application of diverse BAC transgenic rodent lines has enabled rapid progress for precise molecular targeting of genetically-defined cell types in the mammalian central nervous system. These transgenic tools have played a central role in the optogenetic revolution in neuroscience. Indeed, an overwhelming proportion of studies in this field have made use of BAC transgenic Cre driver lines to achieve targeted expression of optogenetic probes in the brain. In addition, several BAC transgenic mouse lines have been established for direct cell-type specific expression of Channelrhodopsin-2 (ChR2). While the benefits of these new tools largely outweigh any accompanying challenges, many available BAC transgenic lines may suffer from confounds due in part to increased gene dosage of one or more “extra” genes contained within the large BAC DNA sequences. Here we discuss this under-appreciated issue and propose strategies for developing the next generation of BAC transgenic lines that are devoid of extra genes. Furthermore, we provide evidence that these strategies are simple, reproducible, and do not disrupt the intended cell-type specific transgene expression patterns for several distinct BAC clones. These strategies may be widely implemented for improved BAC transgenesis across diverse disciplines.

Highlights

Bacterial Artificial Chromosomes (BACs) are large DNA constructs composed of a small cloning vector backbone ligated to large fragments of restriction-digested genomic DNA that can be stably propagated as well as manipulated in bacterial host cells
Our general strategy has been to select BAC clones that have previously been validated in the GENSAT project, as determined by the successful development of mouse lines for cell type-specific expression of Cre or EGFP
With this strategy we have observed that independent BAC transgenic mouse lines created by modification of the same original BAC clone exhibit highly reproducible cell-type specific transgene expression that recapitulates endogenous gene expression patterns

Summary

Introduction

Bacterial Artificial Chromosomes (BACs) are large DNA constructs composed of a small cloning vector backbone ligated to large fragments of restriction-digested genomic DNA that can be stably propagated as well as manipulated in bacterial host cells. Extensive BAC libraries have been constructed with genomic material from a variety of organisms and have served as indispensable tools for large-scale genome sequencing and mapping efforts One such project culminated in the release of three landmark mouse BAC libraries derived from the C57BL/6J and 129S6/SvEvTac strains with a combined 30-fold coverage of the mouse genome (Osoegawa et al, 2000). This resource was quickly tapped to create the first BAC transgenic mouse line with functional transgene expression driven by successful integration of an engineered Bacterial Artificial Chromosome into the mouse genome (Yang et al, 1997). This work boldly asserted the incredible potential of BACs for gene therapy, disease modeling, and other basic research applications aimed at deciphering gene function

Methods

Results

Conclusion