Fluorescence-activated cell sorting of EGFP-labeled neural crest cells from murine embryonic craniofacial tissue.
During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt 1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt 1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.
Singh
Saurabh
S
Department of Molecular, Cellular and Craniofacial Biology, Birth Defects Center, University of Louisville, 501 South Preston Street, Louisville, KY 40292, USA.
Bhattacherjee
Vasker
V
Mukhopadhyay
Partha
P
Worth
Christopher A
CA
Wellhausen
Samuel R
SR
Warner
Courtney P
CP
Greene
Robert M
RM
Pisano
M Michele
MM