Transgene Release DNA Preparation

The transgene is released from vector sequences by restriction endonuclease (REN) digestion. Removal of prokaryotic sequences should be carried out as completely as possible (see Note 1). Depending on the materials available, several methods can be pursued to extract the linearized transgene from agarose gels acid phenol extraction (see Subheading 3.1.2.), agarose treatment (see Note 6), and electroelution (see Subheading 3.1.3.). Electroelution is performed by trapping DNA onto a dialysis...

Protein Tau and FTDP Neuropathology

The most direct indication of the pathophysiology caused by protein tau is the mutations in the tau gene that are linked to neurodegeneration and dementia, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (2). In vitro, most clinical missense mutations located in the microtubule binding domains reduce the association of protein tau with microtubuli (21). In addition, several intronic mutations as well as two missense mutations induce a preponderance of...

Prominent Axonopathy in Tau Transgenic Mice

Three founder transgenic strains, i.e., htau40-1, htau40-2, and htau40-5, with different expression levels of four-repeat human tau were selected, transmitting the transgene stably and in a mendelian fashion. All strains expressed human protein tau exclusively in neurons, with the highest expression in strain htau40-1. Rather unexpectedly, the neuronal excess of human protein tau caused an axonopathy evidenced by proximal axonal dilations with accumulation of neurofilaments, microtubuli,...

Mouse Human Differences

Differences between mouse and human are more obvious to the physician than to the molecular biologist. To the molecular biologists, it is remarkable that mice are so different, since virtually all genes are conserved between mouse and human. Despite differences at the DNA sequence level, it is in most cases possible to substitute entire gene regions and observe no differences in gene regulation and gene function. The same holds true when examining, for example, lipoprotein metabolism. From a...

Endogenous Regulatory Elements Transgene Size

The choice of regulatory elements that drive transgene expression is broad (Fig. 2) and is primarily determined by the aim of the model. However, in all instances, a number of indispensable elements that control gene expression need to be included in a transgene. The promoter, the region of DNA at which gene expression is initiated by binding of the RNA polymerase transcriptional machinery, is the most basic and essential element controlling gene expression. The promoter region should comprise...

Mouse Models in Biomedical Research

The five concluding chapters (Chapters 14-18) provide insight into the work that needs to be done, after the mouse model has been generated. Chapter 14 provides an overview of the pathology studies that should be completed with every new mouse strain generated. Often, transgenic mice are only examined for the presence of predicted phenotypes. It is important, however, to asses the complete mouse for abnormalities. Although one could not apply all possible phenotyping methods to every new mouse...

Thymectomy

Development and maturation of T-cells is dependent on the thymus. Therefore, removal of the thymus creates a mouse devoid of T-cells and can be used to study the role of these cells in immune response. However, this is only true when thymectomy is performed in neonatal mice from birth to day 3. Thymectomy in the adult mouse (> 3 weeks) does not create a fully T-cell-depleted mouse but merely prevents generation of any new T-cells. In these mice, T-cells generated until the time of thymectomy...

Dian Soewarto Vronique Blanquet and Martin Hrabe de Angelis 1 Introduction

Mouse models play an important role in the elucidation of molecular pathways underlying human disease. Mutations in mouse can be generated by a variety of techniques including those using inducing agents such ionizing radiation or chemicals and those that involve genetic manipulations such as transgenic insertions or knockouts. Random mutagenesis by ionizing radiation or chemical agents has a long tradition in classical genetics and has allowed the generation of a large number of mutant...

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...