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Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs
Published on: May 18, 2022
A transient transgenic RNAi strategy for rapid characterization of gene function during embryonic development
Bryan C Bjork1, Yuko Fujiwara, Shannon W Davis
1Genetics Division, Brigham & Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
Plos One
|December 24, 2010
Summary
RNA interference (RNAi) offers a rapid method for studying gene function during embryonic development. The PiggyBac transposon system effectively generates transgenic mouse embryos for developmental RNAi screening.
Area of Science:
- Developmental Biology
- Genetics
- Molecular Biology
Background:
- RNA interference (RNAi) is crucial for understanding gene function by reducing gene expression.
- Characterizing developmental effects of gene dysregulation requires efficient screening methods.
Purpose of the Study:
- To develop a rapid RNAi method for assessing developmental consequences of gene dysregulation in mouse embryos.
- To evaluate lentiviral and transposable element systems for transient transgenic knockdown.
Main Methods:
- Transient transgenic knockdown of mRNA in mouse embryos using lentiviral infection.
- Utilizing Sleeping Beauty (SB) and PiggyBac (PB) transposable element systems for gene knockdown.
- Phenotypic validation of gene targets, including Prdm16, in relation to cleft palate.
Main Results:
- The PiggyBac (PB) transposon system yielded a high number of transgenic embryos with expected phenotypes.
- Knockdown of Prdm16 phenocopied the cleft palate (CP) mutant, csp1, validating the approach.
- Transient transgenic RNAi proved simpler and faster than traditional gene targeting.
Conclusions:
- The PiggyBac transposon system is a powerful tool for high-throughput screening of developmental gene function.
- Transient transgenic RNAi in mouse embryos offers a rapid and effective alternative to embryonic stem cell-based gene targeting.
- This method facilitates rapid phenotypic validation of potential gene targets in development.
Related Concept Videos
02:15Experimental RNAi
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...
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...
01:16In-vitro Mutagenesis
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.