Figure 2. Conditional inactivation of the Grb2 gene in mice.

(A) Schematic representation of the Grb2 locus targeting strategy and the resulting conditional Grb2^flx allele. LoxP sites are represented with white triangles and FRT sites with white circles. Genotyping primers P1 to P4 are shown. (B) Example of 2 positive ES clones targeted at the Grb2 locus, as judged from a positive P1/P2 PCR product of 1200 bp. (C) Example of a successful FLPe-mediated excision of the SA-IRES-βgeo-pA cassette to generate the Grb2^flx allele, as judged from a positive P3/P4 PCR product of 200 bp. (D) PCR analysis of Cre-mediated excision of the Grb2^flx allele in mouse glomeruli. A 235 bp P1/P4 PCR product confirms excision at the locus and correlates with the presence of Cre recombinase. (E) qPCR analysis of Cre-mediated excision of the Grb2^flx allele in FACS-sorted podocytes from Podocin-Cre; Nephrin-CFP; Grb2flx/flx (mutant, Cre+, n = 2) or Nephrin-CFP; Grb2flx/flx (control, Cre-, n = 3) mice. Amplification levels of a P3/P4 PCR product were normalized to B-actin and used to calculate relative copy numbers of the non-excised Grb2 flx allele (Cre-: 2.00±0.37 and Cre+: 0.13±0.01). Star represents p-value of 3.8E-05. (F) Western blot showing 2 examples of ROSA-CreER-mediated inactivation of the Grb2^flx allele in MEFs, resulting in the absence of the Grb2 protein product. Positive Cre (+) indicates treatment with OHT to activate the expression of the transgene.