Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Alternative approach to generate shRNA from cDNA.

Anh Dinh1, Yin-Yuan Mo

  • 1Southern Illinois University School of Medicine, Springfield, IL 62794, USA.

Biotechniques
|May 12, 2005
PubMed
Summary

This study presents a simplified method for generating short hairpin RNA (shRNA) from complementary DNA (cDNA). This new technique enables efficient creation of shRNA libraries for genome-wide gene silencing screens.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Resolution of PML after Treatment with Virus-Specific T Cells and HCT.

The New England journal of medicine·2026
Same author

Intra-apheresis CD34+ cell count: A dynamic approach to predicting peripheral blood stem cell collection yield.

Transfusion·2026
Same author

Stability testing of HPCs and MNCs from apheresis products.

Immunobiology·2025
Same author

RNA binding protein-mediated competing endogenous RNA mechanism in cancer.

Gene·2025
Same author

Correction: Lnc-DC promotes estrogen independent growth and tamoxifen resistance in breast cancer.

Cell death & disease·2025
Same author

An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.

Cell reports. Medicine·2024

Area of Science:

  • Molecular Biology
  • Genetics

Background:

  • Vector-based expression of short hairpin RNA (shRNA) is effective for gene silencing, comparable to in vitro synthesized short interfering RNA (siRNA).
  • Recent advancements allow shRNA library construction from complementary DNA (cDNA), offering potential for large-scale functional genomic screens.

Purpose of the Study:

  • To develop and validate an alternative, simplified method for generating shRNA from cDNA.
  • To demonstrate the utility of this method for creating shRNA libraries for gene function studies.

Main Methods:

  • Utilized a nicking enzyme to create a single-stranded loop in double-stranded DNA at elevated temperatures.
  • Employed Taq DNA polymerase to extend the single-stranded region, forming a palindromic shRNA precursor with a loop.
  • Cloned the resulting DNA fragment into a vector with an H1 promoter and a Pol III terminator (5Ts).
  • Validated the approach by constructing shRNA targeting green fluorescent protein (GFP) and confirming gene expression suppression.

Main Results:

  • Successfully generated shRNA from GFP cDNA using the novel method.
  • Demonstrated significant suppression of GFP expression, validating the efficacy of the generated shRNA.
  • The simplified approach facilitates the creation of shRNA libraries from cDNA.

Conclusions:

  • The developed method offers a simplified and effective alternative for generating shRNA from cDNA.
  • The generated shRNA libraries are valuable tools for identifying siRNA target sequences and conducting genome-wide functional screens.
  • This technique enhances the potential for studying gene functions across various biological systems.

Related Experiment Videos