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Fig. 1. Generation of ß1-integrin null neurosphere cultures from the forebrain of neonatal mice. (A) Schematic representation of the conditional ß1-integrin allele (lox) and Cre recombinase driven recombination leading to the recombined null (
) allele. Exons are indicated by purple boxes, loxP sites by black triangles. Note the promoterless lacZ gene trailing the conditional allele with a splice acceptor site (SA) derived from the intron upstream of exon 2 preceding it. This leads to the expression of ß-gal in recombined cells under the control of the endogenous ß1-integrin promoter. (B) Schematic drawing of the experimental set-up used in this work. Primary neurospheres (2a) were obtained from neonatal forebrains of conditional ß1-integrin null mice. Neurospheres were trypsinized and the single cell suspension exposed to an adenovirus expressing the Cre recombinase (Ad-Cre; panel 2b). After an additional 10 days, primary infected neurospheres were harvested (3a) and either stained for X-gal or passaged (3b) to obtain further generations of infected neurospheres (4). (C) After infection with adeno Cre virus, the expression of ß-gal, monitored by X-gal staining, indicates the recombination of the ß1-integrin flox allele. Note that over 95% of the neurospheres are ß-gal-positive. (D) 12 µm cryostat section of control wild-type neurospheres stained for ß1-integrin. (E-G) Confocal microscopy analysis of 12 µm cryostat sections of recombined lox/wt spheres shows colocalization of ß-gal (reporting ß1-integrin promoter activity; green in E) and nestin (red in F) as yellow in G. Note that ß1-integrin staining shown in D is topographically similar to that of ß-gal in recombined lox/wt neurospheres. Bars, 150 µm (C); 50 µm (D-G).
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