Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells.
This study is the first to report a unique genetic strategy to permanently label mammalian neural crest cells (NCC) with a fluorescent marker, selectively isolate the labeled NCC or their derivatives during murine ontogenesis by laser capture microdissection (LCM), and prepare molecular components, such as RNA, for selective gene expression analyses. Through utilization of a Cre recombinase/loxP system, a genetic strategy that has been used repeatedly to achieve tissue-specific activation of reporter transgenes in mice, a novel two-component mouse model was created in which neural crest cells (and their progeny) are indelibly marked throughout the pre- and postnatal lifespan of the organism. To generate this mouse model, a Wnt1-Cre transgenic line was crossed with a mouse line expressing a conditional reporter transgene ("floxed" enhanced green fluorescent protein). Resulting offspring, expressing both the Wnt1-Cre and "floxed" EGFP alleles, demonstrated EGFP expression in the NCC and all of their derivatives throughout embryonic, postnatal, and adult stages. In the present study, EGFP-labeled cranial NCC from the first branchial arch of gestational day 9.5 murine embryos were visualized in frozen tissue sections and isolated by LCM under epifluorescence optics. RNA was extracted from "captured" cells and amplified by double-stranded cDNA synthesis and in vitro transcription. Amplified mRNA samples from "captured" cells were evaluated by TaqMan quantitative, real-time PCR for the expression of a panel of NCC gene markers. The molecular genetic strategy delineated in this report will facilitate future embryo-genomic and -proteomic analyses of mammalian NCC that will serve to further our understanding of these pluripotent embryonic progenitor cells.
Bhattacherjee
Vasker
V
University of Louisville Birth Defects Center, Department of Molecular, Cellular and Craniofacial Biology, ULSD, Louisville, Kentucky 40292, USA. vasker.bhattacherjee@louisville.edu
Mukhopadhyay
Partha
P
Singh
Saurabh
S
Roberts
Emily A
EA
Hackmiller
Rita C
RC
Greene
Robert M
RM
Pisano
M Michele
MM
D001483:Base Sequence; D017931:DNA Primers; D004622:Embryo, Mammalian / Q000166:cytology*; D004625:Embryo, Nonmammalian; D005456:Fluorescent Dyes / Q000737:chemistry*; D007834:Lasers; D009432:Neural Crest / Q000166:cytology*; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D005456:Fluorescent Dyes