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Related Experiment Videos

Functional genomics in mice by tagged sequence mutagenesis

G G Hicks1, E G Shi, X M Li

  • 1Department of Microbiology and immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2363, USA.

Nature Genetics
|August 1, 1997
PubMed
Summary

A novel gene trap retrovirus vector enables large-scale functional studies by disrupting genes in mouse embryonic stem cells. This method generates promoter-proximal sequence tags (PSTs) for gene identification and functional analysis in vivo.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Developmental Biology

Background:

  • Mammalian gene characterization is advancing, yet functional information remains limited for many cDNA sequences.
  • Large-scale functional genomics requires efficient methods to create and analyze gene disruptions.
  • Murine embryonic stem (ES) cells offer a powerful system for genetic manipulation and studying gene function during development.

Purpose of the Study:

  • To develop a gene trap retrovirus shuttle vector for large-scale gene disruption in ES cells.
  • To create a library of mutant clones for functional genomic studies.
  • To generate promoter-proximal sequence tags (PSTs) for gene identification and characterization.

Main Methods:

  • Development of a gene trap retrovirus shuttle vector.

Related Experiment Videos

  • Disruption of genes in murine embryonic stem (ES) cells using the retrovirus vector.
  • Isolation of mutant clones and sequencing of genomic DNA flanking provirus inserts.
  • Identification and analysis of promoter-proximal sequence tags (PSTs).

    • Main Results:

    • A library of mutant ES cell clones was generated with 400 independent provirus inserts.
    • Sequencing of flanking genomic DNA identified 63 specific genes and anonymous cDNAs disrupted by the retrovirus.
    • The efficiency of tagged sequence mutagenesis indicates broad applicability to thousands of ES cell genes.
    • PSTs were successfully generated as expressed sequence tags from genomic DNA, revealing exon boundaries and promoters.

    Conclusions:

    • The developed gene trap vector is efficient for targeting a large proportion of genes expressed in ES cells.
    • This approach provides defined mutations for in vivo gene function analysis.
    • PSTs derived from genomic DNA offer valuable information on gene features absent in cDNA sequences, advancing functional genomics.