Loss of Function Experiments

Techniques are now available by which specific cell types can be ablated in transgenic animals. This powerful tool has been developed through the use of cytotoxic genes such as the A subunit of diphtheria toxin (DT-A) or ricin, which may be linked in transgene constructs to cell-specific regulatory elements (24,25). Cells expressing such constructs in transgenics are killed. Although the possibilities of this tech nique are exciting, a number of problems may limit its application in studies of the brain. Embryo lethality resulting from toxin expression in cells essential for embryo survival is clearly a potential hazard; even where tissue-specific promoters are used, minimal ectopic expression may be sufficient to result in death caused by the potent nature of these toxins. The recent development of an attenuated DT-A gene (26), which requires a higher level of expression to produce a lethal effect, may prove to be a more versatile tool in genetic ablation experiments. Another problem with these studies regards the possibility that early ablation of one cell type may lead to abnormal development of other cells through the absence of cell interactions. Specificity of ablation may not be obtained and hence any loss of function would be uninterpretable. The use of an inducible toxic gene, such as tk from herpes simplex virus in transgene constructs (27), may circumvent the developmental side effects of cell ablation since transgene expression may be induced at later stages of development. However, since only dividing cells can be killed (27), this approach is of limited use in the brain. A third problem with the ablation technique is that a residual number of cells has generally been shown to escape ablation, thus confusing interpretation. Despite these drawbacks, further application of the cell ablation technique is anticipated. A transgenic mouse model of demyelinating disease has recently been produced (28) using a myelin basic protein (MBP)/DT-A construct (see Section 6.1.). Techniques for disrupting the expression of specific endogenous genes are also available. One promising technique involves the use of constructs designed to express antisense (m)RNA. In the only reported success to date (29), expression of MBP antisense (m)RNA in the brains of transgenic mice reduced levels of both MBP mRNA and MBP, resulting in the shiverer mutant phenotype. An alternative procedure in which specific genes are targeted and disrupted through a homologous recombination event is technically more difficult to achieve, but is potentially more reliable than the antisense construct approach. In the former technique, embryo-derived stem (ES) cells are transformed with a targeting vector containing a modified form of the targeted gene that is functionally impaired. A series of selection procedures are then undertaken to enrich for cells in which the rare event of homologous recombination has occurred. Following the identification of a suitable cell line, blastocysts are microinjected with these cells and chimeric mice are generated. Through selective breeding procedures, with reference to coat color, it is possible to generate hetero- and homozygous animals that exhibit the mutated genotype. A recent experiment of this type has successfully disrupted the int-1 gene, producing a homozygous mouse with a severe ataxic phenotype associated with major defects in midbrain and cerebellar development (30). Previous suggestions that int-1 was involved in brain development (see ref. 30) have, therefore, been dramatically validated. Although the tools for performing these experiments are available for mice (see Chapters 23 and 24), the development of ES cell lines has yet to be achieved for rats.

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