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Manipulating gene expression and signaling activity in cultured mouse limb bud cells
Author(s) -
Lewandowski Jordan P.,
Pursell Taylor A.,
Rabinowitz Adam H.,
Vokes Steven A.
Publication year - 2014
Publication title -
developmental dynamics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.634
H-Index - 141
eISSN - 1097-0177
pISSN - 1058-8388
DOI - 10.1002/dvdy.24128
Subject(s) - limb bud , biology , microbiology and biotechnology , limb development , embryonic stem cell , zone of polarizing activity , reporter gene , apical ectodermal ridge , chondrogenesis , gene , population , gene expression , genetics , embryo , stem cell , mesoderm , demography , sociology
Background: 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. Results: 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. Conclusions: 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. Developmental Dynamics 243:928–936, 2014 . © 2014 Wiley Periodicals, Inc.