Strain Differences

One important prerequisite for effective mutagenesis screens is the choice of appropriate mouse strains. It is known that certain outbred and inbred strains have a different sensitivity to chemical mutagens, presumably owing to a variable capability for DNA repair in their germ cells (29,30). If an outbred strain is selected for a mutagenesis experiment (9,11,20), one has to consider the mixed genetic background, which may complicate further analysis of the mutant phenotype. When inbred strains...

Purification of Adenoviral Vectors

The crude lysate obtained after freeze-thawing is subject to two rounds of centrifugation over CsCl gradients. After the second centrifugation, the virus-containing band is isolated, and CsCl is removed by dialysis. In the final dialysis step, the buffer contains 5 sucrose, which is necessary for virus stability at -80 C. 1. Add 2 mL of CsCl (d 1.45 g cm3) to a clear ultracentrifugation tube (Beckmann, cat. no. 344059) and overlay with 4 mL of CsCl (d 1.2 g cm3). 2. Carefully overlay the CsCl...

Constitutive Recombinase Expression

Cre transgenic mice can be produced through either the pronuclear microinjection of randomly integrating transgene constructs or the targeted introduction of the Cre gene in frame with the start codon of an endogenous gene. The first approach is more straightforward, considering the effort involved in vector construction and its introduction into the germline. However, an appropriate promoter region tested for transgenic expression is required (see Note 5). Since the level and pattern of...