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).