Sanger Institute Gene Trap Resource (SIGTR)

The Sanger Institute Gene Trap Resource was a major project that isolated and characterised gene trap mouse embryonic stem (ES) cell lines to generate reporter-tagged, loss of function mutations.

The project produced more than 10,000 characterised gene trap insertions in 129P2 ES cells that are stored frozen and are freely available to the research community. These lines can be requested from the Mutant Mouse Regional Resource Center (MMRRC) at UC Davis in California on a cost-recovery basis and free of restrictions.

[Genome Research Limited]

Background

As a member of the International Gene Trap Consortium (IGTC), the Sanger Institute Gene Trap Resource (SIGTR) project accelerated the pace of research in mouse genetics by using gene trap vector designs that were equipped with site-specific recombination sites for post-insertional modification of the trapped locus to create other desired alleles.

Vector Information

The vector designs used in creating the SIGTR are shown below. Each vector was engineered in each of the three reading frames designated by the number in the vector name. For example, pGT0lxf represents the frame "0" vector in which the exon upstream of the insertion donates a complete codon. Frame 1 and frame 2 vectors donate 1 and 2 bases, respectively.

For more detailed information on a particular vector, follow the links below to access the full sequence of the vector in FASTA format, the annotated sequence or a restriction map of each vector.

The loxP and FRT sites contained in the vector enabled post-insertional modification of the trapped locus.

Key
Intron: 1.5 kb of Mouse En2 intron 1
SA: splice acceptor of mouse En2 exon 2
β-geo: fusion of β-galactosidase and neomycin transferase
pA: SV40 polyadenylation signal

pGT0lxf

Fasta, Annotated sequence, Map

pGT01xr

Fasta, Annotated sequence, Map

Sequence processing

Sequence tags for the trapped genes were generated by direct sequencing of cDNA generated by 5'RACE. Trace files were generated using ABI3730 capillary sequencers, base-called using the Phred algorithm, and then run through Asp where quality sequence was marked using cut-off values established by examining minimum values required for high-confidence annotation matches.

Automated vector-trimming was performed and the reverse complement of the resulting sequences (sense strand) was passed on to the annotation pipeline. All high-quality sequence tags have been deposited in the Genome Survey Sequence database of GenBank.

Annotation pipeline

In order to assign relevant genetic information to mutated cell lines, the SIGTR employed an annotation pipeline based on high-confidence homology matching of sequences generated by 5' RACE to genomic loci. The positional information was then used to link any genetic information contained in the Ensembl Mouse Database associated with the genomic coordinates. For sequences that could not be resolved using homology to the mouse genome, BLAST was used to query the RefSeq database for homology to protein cDNA.

Handling cell lines

We strongly recommend confirming the cell line upon receipt. The General Information pdf document below contains details on molecular confirmation of the insertion event and culturing the cells. If you believe that the cell line you received is not the cell line you requested, please contact the MMRRC immediately.

If you follow the protocols but find that the cells don't look like ES cells, don't panic. Feeder-free ES cells look different to ES cells grown on feeders. If you are unfamiliar with handling feeder-free ES cells, culture the cells on feeders using the standard protocols.

Protocols

Full description for handling SIGTR cell line requests with embedded protocols.

General Information pdf document

Media

Media preparation for tissue culture.

Media preparation pdf document

ES Cell Culture

Protocols used in the culture of SIGTR cell lines. Includes thawing, expansion, and freezing protocols.

ES Cell Culture pdf document

RT-PCR

Protocol for RT-PCR confirmation of gene trap cell lines and also useful for genotyping gene trap mice.

RT_PCR pdf document

X-Gal Staining

Reagent preparation and protocol for detecting β-glactocidase reporter expression in cells, embryos and tissues.

X-Gal Staining pdf document

Dot Blot Hybridization

Protocol for genotyping gene trap mice that can be used for any gene trap insertion.

Dot-Blot Hypbridization pdf document

Data access

Data on all publicly available gene trap lines (including SIGTR lines) are accessible from the International Gene Trap Consortium's Data access page.

The page offers the following search options:

Order resources

To request a cell line please go to MMRRC Catalog Search Form for SIGTR. This is a form for searching the SIGTR resource at MMRRC, and then requesting specific cell lines

All enquiries about SIGTR cell lines should go to UC Davis. The email address for enquiries is mmrrc@ucdavis.edu.

Useful links

Distribution of SIGTR cell lines via the MMRRC

Gene Trap Resources

  • BayGenomics
  • CMHD - Center for Modelling Human Disease
  • GGTC - German Gene Trap Consortium
  • IGTC - International Gene Trap Consortium
  • TIGEM - Telethon Institute of Genetics and Medicine: Transgenic and Knock-Out Mouse Core Facility (TMCF)

Local Resources

Other Resources

  • GO - Gene Ontology Consortium
  • MGI - Mouse Genomics Informatics at the Jackson Institute
  • NCBI - National Center for Biotechnology Information
  • NCBI Mouse Genome Resources - Information page
  • dbGSS - NCBI Genome Survey Sequences
* quick link - http://q.sanger.ac.uk/hfdr57rh