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Related Experiment Videos

Gene targeting in normal and amplified cell lines.

H Zheng1, J H Wilson

  • 1Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.

Nature
|March 8, 1990
PubMed
Summary

Mammalian gene targeting efficiency is not limited by target DNA concentration. Studies show similar rates in normal and amplified cells, suggesting homology search is not rate-limiting.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Targeted DNA recombination is less efficient in mammalian cells compared to yeast.
  • This difference may be due to the lower concentration of target DNA sequences in mammalian genomes.

Purpose of the Study:

  • To investigate if target DNA concentration limits gene targeting efficiency in mammalian cells.
  • To compare gene targeting rates in Chinese Hamster Ovary (CHO) cells with varying dihydrofolate reductase (DHFR) gene copy numbers.

Main Methods:

  • Utilized a positive/negative selection protocol to assess gene targeting.
  • Compared gene targeting efficiency in normal CHO cells (2 DHFR copies) and amplified CHOC 400 cells (800 DHFR copies).

Main Results:

  • Gene targeting efficiency into the DHFR locus was found to be indistinguishable between normal and amplified CHO cells.
  • The frequency of targeted recombination did not increase with a higher concentration of target DNA.

Conclusions:

  • The concentration of target DNA is not a rate-limiting factor in mammalian gene targeting.
  • The search for homology is not the bottleneck in mammalian gene targeting pathways.
  • Differences in genome size do not explain the disparity in targeting outcomes between yeast and mammalian cells.

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