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Related Experiment Video

Updated: Jan 4, 2026

Development of a Negative Selectable Marker for Entamoeba histolytica
06:05

Development of a Negative Selectable Marker for Entamoeba histolytica

Published on: December 12, 2010

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Split selectable markers.

Nathaniel Jillette1, Menghan Du1,2, Jacqueline Jufen Zhu1

  • 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, 06032, USA.

Nature Communications
|November 2, 2019
PubMed
Summary
This summary is machine-generated.

Researchers developed split selectable markers for efficiently selecting multiple engineered genes. This novel system expands the toolkit for genetic engineering, enabling precise selection in both lentivirus transgenesis and CRISPR gene editing applications.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Selectable markers are crucial for identifying genetically modified cells but their limited variety restricts complex engineering.
  • Current methods often struggle with selecting multiple independent genetic modifications simultaneously.

Purpose of the Study:

  • To develop novel split selectable markers for overcoming limitations in selecting multiple transgenes.
  • To enable efficient selection of cells with desired genotypes in lentivirus-mediated transgenesis and CRISPR-Cas-mediated genome editing.

Main Methods:

  • Designed split selectable marker gene segments fused to inteins for protein trans-splicing.
  • Co-segregated split marker segments with different transgenic vectors (lentiviral and CRISPR).
  • Validated 2-split, 3-split, and 6-split resistance genes (Hygromycin, Puromycin, Neomycin, Blasticidin) and fluorescent proteins (mScarlet).

Main Results:

  • Successfully reconstituted functional marker proteins through intein-mediated protein trans-splicing in host cells.
  • Demonstrated the selection of multiple unlinked transgenes using the split marker system.
  • Validated the system for selecting biallelically engineered cells following CRISPR gene editing.

Conclusions:

  • Split selectable markers offer a versatile solution for selecting multiple genetic modifications.
  • The intein-based system significantly expands the available markers for advanced gene editing and transgenesis.
  • Future development holds potential for selecting an even greater number of genetic modifications in target cells.