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Gotoh et al. 10.1073/pnas.0407577101. |
Fig. 6. Frs2a gene targeting. (A) Schematic of gene targeting for generation of the Frs2a 2F and Frs2a 4F alleles. Restriction sites are shown by letter codes. Exons are indicated by black boxes. Top line shows the wild-type Frs2a gene. The 5' and 3' probes used for Southern blotting are indicated by the open boxes. The Frs2a 2F and Frs2a 4F targeting constructs are shown on the second and third lines, respectively. TK, thymidine kinase ORF. LoxP sites are indicated by the open triangles and are located on either side of the neo gene. FF and FFFF indicate the location of the tyrosine to phenylalanine mutations generated in each targeting vector. (B) Southern blotting showing the change in restriction fragment size appropriate for targeting of the 2F and 4F alleles with a BglII digest probed with either the 5' probe (Left) or the 3' probe (Right). (C) PCR amplification of wild-type, heterozygote, and homozygous mutant genomic DNA using the primers shown in A that amplify across the loxP site that remains after excision of the neo gene. (D) Immunoblot analyses of FRS2a with anti-FRS2a antibodies (Upper) and Shp2 (Lower) with anti-Shp2 antibodies in lysates from 2F and 4F mutant cells.
Table 1. The proportion of embryos of a given genotype at different developmental stages for the Frs2a 2F line
|
Embryonic stage |
Genotype |
||
|
WT |
Frs2a +/2F |
Frs2a 2F/2F |
|
|
E9.5 |
26 |
33 |
12 |
|
% |
36.6 |
46.5 |
16.9 |
|
E14.5 |
18 |
30 |
6 |
|
% |
33.3 |
55.6 |
11.1 |
|
E15.5 |
14 |
36 |
7 |
|
% |
24.6 |
63.2 |
12.3 |
|
E17.5 |
10 |
40 |
5 |
|
% |
18.2 |
72.7 |
9.1 |
Supporting Text
Generation of Frs2a 2F and Frs2a 4F mutant mice
The tyrosine codons that correspond to either Shp2-binding sites or the Grb2-binding sites were mutated to encode for phenylalanine residues in a genomic clone of the Frs2a gene (Fig. 1A) and the 2F and 4F genomic clones were then used to build targeting constructs (Fig. 1A). Gene targeting was carried out using standard techniques to obtain Frs2a 2F/neo or Frs2a 4F/neo mice. These mice were crossed with transgenic mice expressing germ-line Cre recombinase (1) to remove the neo cassette. Correct targeting and removal of the neo cassette was confirmed by Southern blot analyses by using specific probes (Fig. 1B). The neo- mice were genotyped by PCR (Fig. 1C) and were used for ongoing analysis. The level of expression of mutant proteins was shown to be similar to the expression of wild-type FRS2a as revealed by immunoprecipitation and immunoblotting analyses of lysates prepared from whole embryonic day (E)14.5 homozygous mutant embryos using anti-FRS2a antibodies (Fig. 1D Upper). Immunoprecipitation and immunoblotting of Shp2 using anti-Shp2 antibodies of the same lysates served as a positive control (Fig. 1D Lower).
1. White, J. K., Auerbach, W., Duyao, M. P., Vonsattel, J. P., Gusella, J. F., Joyner, A. L. & MacDonald, M. E. (1997) Nat. Genet. 17, 404-410.