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.
RT-PCR
Protocol for RT-PCR confirmation of gene trap cell lines and also useful for genotyping gene trap mice.
X-Gal Staining
Reagent preparation and protocol for detecting β-glactocidase reporter expression in cells, embryos and tissues.
Dot Blot Hybridization
Protocol for genotyping gene trap mice that can be used for any gene trap insertion.
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
- MMRRC Background
- MMRRC & SIGTR Cell Lines
- SIGTR Cell Line Catalogue at the MMRC
- Other Mouse Resources Listed at MMRRC
- MTA When Ordering SIGTR Clones via 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
- EMBL-EBI - European Bioinformatics Institute
- Ensembl Mouse Genome Server
- Mouse developmental genetics and ES cell mutagenesis at the Sanger Institute
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
