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Function of PI3Kγ in Thymocyte Development, T Cell Activation, and Neutrophil Migration
Takehiko Sasaki, Junko Irie-Sasaki, Russell G. Jones, Antonio J. Oliveira-dos-Santos, William L. Stanford, Brad Bolon, Andrew Wakeham, Annick Itie, Dennis Bouchard, Ivona Kozieradzki, Nicholas Joza, Tak W. Mak, Pamela S. Ohashi, Akira Suzuki, and Josef M. Penninger

Supplementary Material

Generation of PI3Kγ-Deficient Mice and Gross Phenotype
PI3Kγ-/- mice were generated by replacing 5 kb of the murine p110γ gene (amino acids 1 to 670), including the NH2-terminal region required for the regulation of kinase activity by Gβγ and the putative PH domain, with a PGK-NEO cassette (Web fig. 1A). Southern, Western, and Northern blot analyses confirmed disruption of the gene and the lack of PI3Kγ protein expression in neutrophils, T cells, and thymocytes (Web fig. 1, B and C and not shown, respectively). Expression of p110α, p110β, and p85α was equivalent to that in wild-type littermates (Web fig. 1D). Homozygous PI3Kγ-/- mice were born at the expected Mendelian ratio and appeared healthy. In vitro embryonic stem (ES) cell differentiation into a variety of cell lineages (embryoid bodies, ectoderm, endoderm, mesenchym, beating cardiomyocytes, benzidine-positive blood islands, primitive PECAM-1+ vasculature, and CD45+ hematopoietic cells) was comparable among wild-type, PI3Kγ+/-, and PI3Kγ-/- ES cell lines, indicating that PI3Kγ has no apparent role in germ layer development. Both male and female PI3Kγ-/- mice were fertile. There were no statistically significant differences among PI3Kγ+/+, PI3Kγ+/-, and PI3Kγ-/- littermates in blood levels of glucose, uric acid, cholesterol, triglyceride, phosphorus, calcium, albumin, total protein, total bilirubin, urea nitrogen, creatinine, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, or alkaline phosphatase.

Figure 1. Gene targeting of PI3Kγ in ES cells and PI3Kγ expression. (A) Partial restriction map of genomic PI3Kγ sequences and construction of the neomycin resistance (Neo) insertion vector. PI3Kγ exons are shown as boxes. The PI3K( flanking probe used for Southern blotting and expected fragment sizes after digests of wild-type (6.0 kb) and mutant (3.0 kb) genomic DNA are indicated. E, Eco RI; S, Spe I. (B) Southern blotting. Genomic DNA was isolated from PI3Kγ+/+, PI3Kγ+/-, and PI3Kγ-/- mice; digested with Eco RI, and analyzed by Southern blotting using the 3( flanking probe shown in (A). Wild-type and mutant bands are indicated. (C) Western blot analysis of protein expression of PI3Kγ in neutrophils, thymocytes, and purified lymph node T cells from PI3Kγ+/- (+/-) and PI3Kγ-/- (-/-) mice. Freshly isolated cells were lysed, and 30 (g of lysate was probed with an antibody to PI3Kγ (p110γ). (D) Western blot analysis of protein expression of PI3Ks in neutrophils from PI3Kγ+/- and PI3Kγ-/- mice. Freshly isolated neutrophils were lysed, and 30 μg of lysate was probed with antibodies to PI3Kα (p110α), PI3Kβ (p110β), PI3Kγ (p110γ), and p85α.

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Materials: A 12-kb genomic PI3Kγ fragment was isolated from a 129/J mouse library and inserted into the Not I site of pBluescript II. A targeting vector was constructed containing a 709-base pair (bp) short arm and a 5.4-kb long arm of homology flanking a neomycin resistance cassette (Neo) inserted into the Eco RI/Bgl II sites of the genomic clone in antisense orientation to PI3Kγ transcription. The linearized construct was electroporated into 1 107 E14K ES cells derived from 129/Ola mice. ES cell colonies resistant to G418 (300 μg/ml) were screened for homologous recombination by polymerase chain reaction (PCR) using primers specific for PI3Kγ genomic sequences and Neo (PI3Kγ sense primer: 5(-GGACACGGCTTTGATTACAATC-3(; PI3Kγ antisense primer: 5(-GGGGTGGGATTAGATAAATG-3(). Recombinant colonies were confirmed by Southern blotting of Eco RI- or Spe I-digested genomic DNA hybridized to a 530-bp 3( flanking probe. Eight different targeted ES cells derived from seven independent electroporation experiments were injected into blastocysts from C57BL/6 female mice. Chimeric male mice were crossed with C57BL/6 females to achieve germ line transmission. Following heterozygous matings, PI3KγSUP>-/- mice were distinguished from PI3Kγ+/- and PI3Kγ+/+ mice by PCR genotyping (wild-type primers: sense, 5(-TCAGGCTCGGAGATTAGGTA-3(; antisense, 5(-GCCCAATCGGTGGTAGAACT-3(; mutant primers, as above) and Southern blotting. For Northern blotting, polyA-selected RNA was isolated from PI3Kγ+/+, PI3Kγ+/-, and PI3Kγ-/- lymph node cells, thymocytes, and spleen cells; electrophoresed; transferred to a Hybond-N membrane (Amersham); and hybridized with probes specific for PI3Kγ, Neo, or β-actin. PI3Kγ protein was detected by Western blotting using polyclonal antibodies reactive to the PI3Kγ (p110γ) epitopes amino acids 2 to 17, 1032 to 1050, and 742 to 756 (Santa Cruz Biotechnology). The PI3Kγ mutation was backcrossed onto a C57BL/6 mouse background for three generations. Only littermate mice were used in all experiments. Equivalent results and phenotypes were obtained for two independent PI3Kγ-/- mouse lines derived from two different targeted ES cell clones. All mice were maintained at the animal facilities of the Ontario Cancer Institute under specific pathogen-free conditions according to institutional guidelines.