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Related Concept Videos

In-vitro Mutagenesis01:16

In-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.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

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Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor
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AAV-mediated gene targeting.

Daniel G Miller1

  • 1Department of Pediatrics, University of Washington, Seattle, WA, USA. dgmiller@u.washington.edu

Methods in Molecular Biology (Clifton, N.J.)
|October 29, 2011
PubMed
Summary
This summary is machine-generated.

Adeno-associated virus (AAV) vectors efficiently deliver DNA for homologous recombination in primary cells, improving gene therapies and DNA repair studies. This AAV-mediated gene targeting enables precise genetic modification of cells for research and potential autologous transplantation.

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Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Published on: October 18, 2022

Area of Science:

  • Molecular Biology
  • Gene Therapy
  • Cellular DNA Repair

Background:

  • Homologous recombination is crucial for gene therapies and understanding gene function and DNA repair.
  • Current methods like transfection and electroporation show limited efficiency in delivering DNA to primary cells for homologous recombination.
  • Inefficient nuclear delivery of template DNA hinders the frequency of homologous recombination in primary cells.

Purpose of the Study:

  • To describe a basic approach for gene targeting using adeno-associated virus (AAV) vectors.
  • To review vector design strategies for efficient gene targeting.
  • To present protocols for modifying expressed and non-expressed loci in primary somatic and stem cells.

Main Methods:

  • Utilizing adeno-associated virus (AAV) vectors for efficient delivery of single-stranded DNA recombination templates.
  • Leveraging the AAV genome structure, including single DNA strands stabilized by inverted terminal repeat sequences.
  • Developing protocols for gene targeting in primary somatic cells and stem cells.

Main Results:

  • AAV vectors demonstrate efficient delivery of recombination templates to the nucleus of primary cells.
  • AAV-mediated gene targeting leads to increased homologous recombination frequencies.
  • The strategy allows for the modification of cells from individuals for detailed study.

Conclusions:

  • AAV-mediated gene targeting offers a powerful strategy for precise genetic modification of primary cells.
  • This approach facilitates the study of gene function, DNA repair, and disease mechanisms.
  • The ability to modify cells opens possibilities for autologous transplantation of corrected cells.