TIGM Gene Trap
Technologies 
Overview
The TIGM ES cell clones utilize gene traps for gene
inactivation. In this approach, a promoterlessmarker/reporter
gene (for instance encoding Neo, or βgeo) is
introduced into ES cells. Selection for expression of the gene requires
transcription from a cellular promoter. Consequently, amutation
in a cellular gene and the activity of the tagged gene can be followed by
staining for β-galactosidaseactivity. Unlike
gene targeting by homologous recombination, a single gene trap vector can be
used in a high-throughput fashion to mutate thousands of individual genes in
mouse ES cells, as well as enable the rapid identification of the mutated
genes. Large scale sequencing of ES cell clones was conducted and sequence tags
have been generated. This information was recently submitted to GenBankto serve the research community at large. A list of
the genes we have trapped can be found in our gene trap database. Detailed description of our technology may be found in Wnk1 kinasedeficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention. Proc Natl Acad SciU S A. 2003 Nov
Vector Design
The basic gene trap vectors we have used include a reporter gene downstream
of a splice acceptor sequence (Fig.1 and 2). They are designed to function when
inserted in an intron, to produce incorrect splicing of the target gene such that all exonsdownstream of
the insertion site are not expressed. The gene trap cassette is inserted in a
retroviral vector. Retroviruses insert as a single copy per locus, with no
rearrangement of flanking sequences. They have a preference for insertions at
the 5' end of genes, often upstream of the initiator ATG, and the splice
acceptor sequence we use does not appear to be bypassed by the RNA-splicing
machinery. As a result, the majority of the mutations generated using our gene
trap vectors are predicted to lead to null alleles. Analysis of non-embryonic
lethal mouse lines demonstrates that gene-trap insertions within both exonsand intronsof the gene of
interest lead to the disruption of the endogenous mRNA transcript in all cases.
Of these, >96% show complete absence of WT message, with the remaining hypomorphiclines showing an average reduction in mRNA
levels of 91.6%, as measured by quantitative PCR. These data demonstrate that intragenicinsertion efficiently disrupts gene transcription
in vivo and can be used to reliably predict mutagenicitybefore
mouse production.
TIGM's C57BL/6 ES cell clones were generated using several
different retroviral gene trap vectors that all contain the 5' selectable marker
β-geo, a functional fusion between the β-galactosidaseand
neomycin resistance genes, for identification of successful gene trap events.
The β-geo marker also allows forin vivo
expression studies of the trapped gene through in situ hybridization, immunohistochemistry,
or β-galactosidase-based assays that can detect
the fusion transcript generated as part of the gene trapping process.
Figure 1. Gene trap vectors used in C57BL/6 library *.
We have used high-throughput
gene-trapping with retroviral vectors in mouse C57BL/6 ES cells to generate a
library of more than 300,000 mutated ES cell clones. Each clone is frozen in
duplicate in liquid nitrogen. Wegenerated a tractable
sequence tag from all clones using a third replicate of the ES cell clones that
was subjected to an automated iPCR-based
direct-sequencing protocol. We refer to such sequence as ISTs, which represent
genomic sequence of the mutated genes upstream of the genomic insertion site.
* LTR, long
terminal repeat; PGK, phosphoglyceratekinase-1 promoter; SD, splice donor
sequence; SA, splice acceptor sequence; Neo, neomycin phosphotransferase gene;
β-geo, galactosidase/neomycin phosphotransferase fusion gene; pA,
polyadenylation sequence; Btk, first exon of the murine Bruton's tyrosine kinase gene.
The retroviral vectors (Fig. 2) contain a splice acceptor sequence (SA) followed by
a promoterlessselectable marker Neo with a polyadenylationsignal (pA).
Insertion of the retroviral vector into an expressed gene leads to the splicing
of the endogenous upstream exonsinto this cassette to
generate a fusion transcript. The vectors also contain a promoter that is
active in ES cells [such as that of the mouse phosphoglyceratekinase (Pgk) gene] followed by a first exon (such
as that of the Bruton's Tyrosine Kinase (Btk) gene) upstream of a splice donor (SD)
signal. Splicing from this signal to the exonsdownstream
of the insertion gives rise to a fusion transcript that can be used to generate
a sequence tag (OST) of the trapped gene by 3' RACE ( Zambrowicz, 1998). The Btk exon contains
termination codons in all reading frames to prevent
translation of downstream fusion transcripts.
Figure 2. Gene trap vectors used in 129SvEv library*
High-throughput gene-trapping with retroviral vectors has been used in mouse 129SvEv ES cells
to generate a library of 522,666 mutated ES cell clones. Each clone is frozen
in duplicate in liquid nitrogen. Tractable sequence tags have been generated
from 271,860 clones (52%), using a third replicate of the ES cell clones that
was subjected to an automated RT-PCR-based direct-sequencing protocol (Zambrowicz, 2003).
Such sequences are being referred to as OSTs, which
represent cDNAsequence of the mutated genes
downstream of the genomic insertion site (Fig. 2).
* LTR, long
terminal repeat; PGK, phosphoglyceratekinase-1 promoter; SD, splice donor
sequence; SA, splice acceptor sequence; Neo, neomycin phosphotransferase gene;
β-geo, galactosidase/neomycin phosphotransferase fusion gene; pA,
polyadenylation sequence; Btk, first exon of the murine Bruton's tyrosine kinase gene.