Work

Protocol for the derivation and culture of murine trophoblast organoids for CRISPR-Cas9 screening

Year: 2024

Type: article

Abstract: Murine trophoblast organoids present a more balanced array of trophoblast subtypes, rendering them a suitable platform for CRISPR-Cas9-based screening. Here, we present a protocol for the derivation and culture of murine trophoblast organoids from trophoblast stem cells or placentae. We describe steps for establishing and differentiating murine trophoblast organoids, the characterization of trophoblast organoids in both conditions, the generation of focused single guide RNA (sgRNA) libraries, and the subsequent screening using those libraries in murine trophoblast organoids. For complete details on the use and execution of this protocol, please refer to Mao et al. 1 • Steps for deriving murine trophoblast organoids from placentae or trophoblast stem cells • Culture conditions for long-term passage and preservation of trophoblast organoids • Differentiation condition for replicating placental trophoblast cell types in organoids • Instructions for performing CRISPR-Cas9 screening in murine trophoblast organoids Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Murine trophoblast organoids present a more balanced array of trophoblast subtypes, rendering them a suitable platform for CRISPR-Cas9-based screening. Here, we present a protocol for the derivation and culture of murine trophoblast organoids from trophoblast stem cells or placentae. We describe steps for establishing and differentiating murine trophoblast organoids, the characterization of trophoblast organoids in both conditions, the generation of focused single guide RNA (sgRNA) libraries, and the subsequent screening using those libraries in murine trophoblast organoids.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103405

Authors: Qian Mao, Jingwei Jiang, Qinying Ye, Haopeng Wang, Chao-Po Lin

Institutions: ShanghaiTech University, Shanghai Clinical Research Center

Language: English

Topic: Pluripotent Stem Cells Research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,500