Detection of gene recombination in endoxifen- or Adeno-Cre-induced tumours. Detection of gene recombination in endoxifen- or Adeno-Cre-induced tumours.

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Detection of gene recombination in endoxifen- or Adeno-Cre-induced tumours. Detection of gene recombination in endoxifen- or Adeno-Cre-induced tumours. (A,B) Microdissection of tumours. Tumours correspond to T1 (A) and T2 (B) (see C). (A,B) Prior to microdissection; (A′,B′) after microdissection. (C) Recombination PCR showing a 612-bp band (blue arrowhead) in recombined tumour tissue (T1, T2) but not in wild-type (wt) brain, or non-recombined brain tissue of naïve GLAST::Cre(ERT2);PtenloxP/loxP;p53loxP/loxP mice (lox nr); NT, non-template control; RC, in vitro-recombined cell line derived from the SVZ of a PtenloxP/loxP;p53loxP/loxP mouse. (D) PCR detecting wild-type and non-recombined DNA only. A 512-bp band (red arrowheads) is seen only in non-recombined (lox nr) brain but not in recombined tumour (T1, T2). A 400-bp wild-type is seen in non-transgenic controls only. No signal is detected in the non-template control (NT) or in recombined in vitro-recombined cell line derived from the SVZ of a PtenloxP/loxP;p53loxP/loxP mouse. (E-H) Homozygous PtenloxP/loxP recombination (exon 5) leads to loss of functional PTEN protein and results in upregulation of pAkt (brown immunolabelling). (E,G) Tumour cells that undergo recombination of both alleles express detectable amounts of pAkt, whereas adjacent CNS tissue does not express pAkt (F,H). Scale bar: 100 µm in E-H. Anna Benedykcinska et al. Dis. Model. Mech. 2016;9:211-220 © 2016. Published by The Company of Biologists Ltd