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Many of the MRDDRC projects involve generating mutations
in the germ line and somatic tissues of mice by performing homologous
recombination in Embryonic Stem (ES) cells. The procedures for the manipulation
of ES cells and the construction of mice are capital intensive and technically
demanding. There are many different types of alleles which the Core
will be able to help to establish in the mouse germ line. These alleles
vary in complexity from "simple" knockouts to more sophisticated
alterations generated through successive manipulations of ES cells.
For example, this might include the generation of chromosomal deficiencies,
the generation of conditional alleles such as flanking an exon of a
gene with loxP sites and targeting Cre to specific
loci. All of these allele types have been generated previously by the
Core and therefore this should not present any specific problem. A summary
of the types of alleles which will be generated by the Core is presented
in Figure 1. The Core will be set up to assist
various projects studying mental retardation with the generation of
mice with targeted mutations. Having mouse models for various MRDDRC
disorders is an essential step for studies aimed at understanding pathogenesis
as well as for any investigations involving therapeutic intervention.
Investigators will be able to receive the
assistance of the Core at several phases in their experiments:
1. Construct design, supply of vectors
and genomic libraries
2. Targeting in ES cells
3. Germline transmission of targeted alleles
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Figure
1. Types of mutant alleles which can be generated by
the Core. |
A. |
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A hypothetical three exon
gene with a non-coding first exon. |
B. |
A simple knockout where the Hprt
selection cassette deletes the second exon thereby mutating the
gene. |
C. |
A Cre knockin into the hypothetical
gene. Cre is brought under the transcriptional control
of the target locus by virtue of a splice acceptor site (SA).
This allele also contains Hprt for positive selection,
but this could be removed if necessary. D1 & D2
represent the two stages in the generation of a floxed allele. |
D1. |
In the first step the locus is targeted
using Hprt for positive selection. Hprt is placed
in the intron so that after removal by Cre-excision
the remaining loxP site in the intron is unlikely to
interfere with the function of the locus. |
D2. |
The final locus is similar to the wild
type locus except one of the exons (exon 2) is flanked by
loxP sites. Tissue specific expression of Cre will
delete the DNA flanking these sites resulting in tissue specific
mutation of the gene of interest. E1, E2, & E3 depict the
3 stages in the generation of a deletion allele. |
E1. |
A region of the genome with 4 genes,
V, W, X & Z. |
E2. |
Targeting of the deletion cassettes containing
the portions of the split Hprt minigene cassette to the
deletion end points. |
E3. |
The final product after Cre expression
and HAT selection. In this case the genes W and X have been deleted.
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| Figure
2. The Tbx1lacZ knock-in allele,
obtained with the support of the MRDDRC Core, delineates gene
expression in E10.5 embryos. Note the absence of the second
branchial arch (arrow) in the Tbx1-/- embryo. The coronal
sections below are from similar stage embryos and illustrate
the severity of the abnormalities of the pharyngeal apparatus
in homozygous mutants. |
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