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Related Concept Videos

Experimental RNAi02:15

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RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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MicroRNAs01:22

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MicroRNA (miRNA) are short, regulatory RNA transcribed from introns (non-coding regions of a gene) or intergenic regions (stretches of DNA present between genes). Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself, forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA...
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Related Experiment Video

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Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits
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Efficient Gene Knockdowns in Mouse Embryonic Stem Cells Using MicroRNA-Based shRNAs.

Jianlong Wang1

  • 1Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Mount Sinai School of Medicine, Atran Bldg., AB7-10D, 1428 Madison Avenue, Box 1020, New York, NY, 10029, USA. jianlong.wang@mssm.edu.

Methods in Molecular Biology (Clifton, N.J.)
|July 5, 2017
PubMed
Summary
This summary is machine-generated.

This chapter details protocols for RNA interference (RNAi) gene knockdown in mouse embryonic stem cells (ESCs). It covers methods for efficient gene silencing using microRNA-embedded short-hairpin RNA (shRNAmir) for genetic studies.

Keywords:
Embryonic stem cellsMicroRNAMicroRNA-embedded short-hairpin RNA (shRNAmir)RNA interference

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Area of Science:

  • Molecular Biology
  • Genetics
  • Stem Cell Biology

Background:

  • RNA interference (RNAi) is a key technology for gene knockdown in eukaryotic systems.
  • Embryonic stem cells (ESCs) are crucial for understanding self-renewal, pluripotency, and cell differentiation.
  • RNAi in ESCs aids in dissecting pluripotency mechanisms and developing cell replacement therapies.

Purpose of the Study:

  • To provide detailed protocols for RNA interference (RNAi) studies in mouse ESCs.
  • To enable efficient gene knockdown using microRNA-embedded short-hairpin RNA (shRNAmir).
  • To establish methods for both constitutive and inducible gene silencing.

Main Methods:

  • Utilizing retrovirus infection for integrating shRNAmir cassettes into the genome.
  • Employing loxP site-directed recombination for inducible gene knockdown.
  • Performing RNA interference (RNAi) in mouse ESCs.

Main Results:

  • Established protocols for potent RNAi response in mouse ESCs.
  • Demonstrated efficient gene knockdown via viral integration and site-specific recombination.
  • Enabled both constitutive and inducible gene silencing strategies.

Conclusions:

  • shRNAmir-mediated RNAi is an effective tool for functional genetic studies in ESCs.
  • The provided protocols facilitate detailed investigation of ESC self-renewal and pluripotency.
  • These methods support the advancement of cell replacement therapies through precise gene manipulation.