Protocol to investigate the effects of localized gene expression disruption on mouse limb development via direct siRNA injection and explant culture.

Ji-Hye Yea, Sharon E S Onggo, Suhani Dewagan, Kaiwen Chen, Kaily K Young, Patrick Cahan

Abstract read

In one paragraph

Article in STAR protocols, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.

0numbers the graph read from it

0cells of the map it votes in

0citing papers in PubMed

–field-weighted citation impact

1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

0 citing papers in PubMed.

No citing paper in PubMed yet.

4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

6 authors.

Ji-Hye YeaDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: jyea2@jh.edu.

Sharon E S OnggoDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Suhani DewaganKrieger School of Arts and Sciences, Johns Hopkins University, Baltimore, MD 21218, USA.

Kaiwen ChenDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Kaily K YoungInstitute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Patrick CahanDepartment of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: patrick.cahan@jhmi.edu.

Funding

From intra to intercellular regulatory networks that define cell type identityR35GM124725 · NIGMS · JOHNS HOPKINS UNIVERSITY · PI Patrick Cahan · 2017 to 2026

$4.5M

NIGMS NIH HHS R35 GM124725

6 · The paper itself

Abstract

Here, we present a protocol for localized gene knockdown in embryonic mouse hindlimb explants using direct small interfering RNA (siRNA) injection, combined with a semi-quantitative morphology scoring system to assess developmental outcomes. We describe steps for mating of samples, preparing hindlimb explants for culture and siRNA-Lipofectamine complex, and direct injection of the siRNA-Lipofectamine complex. We then detail procedures for explant culture and maintenance, data analysis, tissue fixation, and cryoprotection. This protocol approach enables spatially precise modulation of gene expression during limb and joint development.

Indexed as

Gene Expression Regulation, DevelopmentalGene Knockdown TechniquesHindlimbRNA, Small InterferingTissue Culture TechniquesAnimalsMiceRNA, Small Interferingdevelopmental biologyStem cellstissue Engineering

Identifiers

PMID41764661

DOI10.1016/j.xpro.2026.104402

PMCPMC12964285

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LicenceCC BY

Read underepoch 390

Registered trials

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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.