Manipulating gene expression and signaling activity in cultured mouse limb bud cells.

The vertebrate limb bud is a well-established system for studying the mechanisms driving growth and patterning of an embryonic tissue. However, approaches for manipulating gene expression are currently limited to time-consuming methods. Culturing primary limb bud cells could potentially be used as a quicker assay. However, limb cells in culture quickly differentiate into cartilage under normal conditions, and approaches delivering DNA and siRNA into primary limb cells in culture are limited. These technical limitations have restricted the utility of limb buds for investigating problems that require higher-throughput approaches. In this report, we describe adaptations to a method for culturing primary limb bud cells in a pre-chondrogenic state, and generate a population of mouse primary limb cells that are responsive to Hedgehog (Hh) signaling. Hh-stimulated cells upregulate Hh target genes as well as an exogenous Hh-responsive reporter. We then describe a method for highly efficient delivery of plasmids and siRNAs into cultured primary limb bud cells in a 96-well format. Cultures of primary limb bud cells are amenable to gene manipulation under conditions that maintain the limb cells in an Hh-responsive, undifferentiated state. This approach provides a medium-throughput system to manipulate gene expression, and test DNA regulatory elements.