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Next-generation Sequencing03:00

Next-generation Sequencing

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Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells
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Published on: January 17, 2019

Cloning and sequencing an inverted repeat.

Petr Svoboda1

  • 1Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. svobodap@img.cas.cz

Cold Spring Harbor Protocols
|February 12, 2010
PubMed
Summary
This summary is machine-generated.

We present three methods for cloning inverted repeats (IRs) for transgenic RNA interference (RNAi) in mice. Sequencing strategies are also discussed to ensure the integrity of these crucial gene silencing constructs.

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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)
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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

Published on: October 5, 2018

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • RNA interference (RNAi) enables sequence-specific post-transcriptional gene silencing.
  • Transgenic RNAi in mice requires cloning an inverted repeat (IR) into the Zp3 transgenic cassette.

Purpose of the Study:

  • To describe successful strategies for cloning inverted repeats (IRs) for transgenic RNAi in mice.
  • To detail methods for sequencing cloned IRs to ensure construct integrity.

Main Methods:

  • Described three distinct cloning strategies: PCR product ligation, sequential cloning with a short spacer, and sequential cloning with a long spacer.
  • Outlined two sequencing approaches: single internal primer for both arms and separate external primers for each arm.

Main Results:

  • Successfully cloned inverted repeats (IRs) using the described ligation and sequential cloning methods.
  • Demonstrated the effectiveness of both internal and external primer strategies for sequencing cloned IRs.

Conclusions:

  • The presented cloning strategies are effective for generating transgenic RNAi constructs in mice.
  • External primer sequencing is recommended for verifying the integrity of cloned inverted repeats in transgenic constructs.