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Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction

Huss, David and Choi, Harry M. T. and Readhead, Carol and Fraser, Scott E. and Pierce, Niles A. and Lansford, Rusty (2015) Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction. Cold Spring Harbor Protocols, 2015 (3). pp. 259-268. ISSN 1940-3402. https://resolver.caltech.edu/CaltechAUTHORS:20150311-133810360

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Abstract

Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1101/pdb.prot083832DOIArticle
http://cshprotocols.cshlp.org/content/2015/3/pdb.prot083832.abstractPublisherArticle
ORCID:
AuthorORCID
Choi, Harry M. T.0000-0002-1530-0773
Fraser, Scott E.0000-0002-5377-0223
Pierce, Niles A.0000-0003-2367-4406
Lansford, Rusty0000-0002-2159-3699
Additional Information:© 2015 Cold Spring Harbor Laboratory Press. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/. This work draws on molecular architectures developed within the NSF Molecular Programming Project (NSF-CCF-0832824 and NSF-CCF-1317694) and was funded by the NIDCR FaceBase Consortium (U01-DE020063), the NIH (5R01EB006192), the Gordon and Betty Moore Foundation (GBMF2809), the Beckman Institute at Caltech (Programmable Molecular Technology Center).
Funders:
Funding AgencyGrant Number
NSFCCF-0832824
NSFCCF-1317694
NIDCR FaceBase ConsortiumU01-DE020063
NIH5R01EB006192
Gordon and Betty Moore FoundationGBMF2809
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:3
Record Number:CaltechAUTHORS:20150311-133810360
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150311-133810360
Official Citation:Combinatorial Analysis of mRNA Expression Patterns in Mouse Embryos Using Hybridization Chain Reaction David Huss, Harry M.T. Choi, Carol Readhead, Scott E. Fraser, Niles A. Pierce, and Rusty Lansford Cold Spring Harb Protoc; 2015; doi:10.1101/pdb.prot083832
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:55707
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:11 Mar 2015 21:21
Last Modified:09 Mar 2020 13:18

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