Gene knock-outs

Biological raw data are stored in public databanks (such as Genbank or EMBL for primary DNA sequences). The data can be submitted and accessed via the World Wide Web. Protein sequence databanks like trEMBL provide the most likely translation of all coding sequences in the EMBL databank. Sequence data are prominent, but also other data are stored, e.g.yeast two-hybrid screens, expression arrays, systematic gene-knock-out experiments, and metabolic pathways. 

Desmoplakin is the most abundant desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque. Desmoplakin forms rod-like dimers that bind to intermediate filaments and to the cadherin-associated proteins plakoglobin and plakophilin. Gene knock-out experiments have revealed an essential role of desmoplakin in establishing cell-cell contacts in early mouse embryos. 

Knockout animal studies, in contrast to the above methods, are dependent upon phenotype observation. The approach entails the generation and study of mice in which a specific gene has been deleted. Phenotypic studies can sometimes yield clues as to the function of the gene knocked out. 

Neurotoxins such as mercaptopyrazide pyrimidine (MPP+) and 6-hydroxydopamine are also taken up by transporters, and this is required for their neurotoxic effects. Mice have been prepared with their transporter genes knocked out . Extensive studies with these mice confirm the important role of transporters (Table 12-1). Once an amine has been taken up across the neuronal membrane, it can be taken up by intracellular adrenergic storage vesicles as described above. 

Heber, S., Herms, J., Gajic, V. et al. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci. 20 7951-7963,2000. 

Wu et al. (1998) noted that doxombicin-induced apoptosis in lymphoid cells was blocked by pepstatin A, which is an inhibitor of cathepsin D. These investigators also observed that cathepsin D was induced through p53 DNA-binding sites at the cathepsin D promoter. Moreover, they have foimd that, compared to fibroblasts from wild-type mice, cathepsin D-/- fibroblasts from gene knock-out mice exhibited increased resistance to death caused by doxombicin. Also, in semm-deprived rat PC 12 cells undergoing apoptosis, the amoimt of cathepsin B has been observed to decline, while the level of cathepsin D increased (Shibata et al, 1998), and, in our laboratory (Kagedal et al, 2001), the same phenomenon was recently seen in human fibroblasts exposed to naphthazarin. 

Harrison GJ, Cerniway RJ, Peart J, Berr SS, Ashton K, Regan S, Matherne GP, Headrick JP (2002) Effects of A3 adenosine receptor activation and gene knock-out in ischemic-reperfusion mouse heart. Cardiovasc Res 53(1) 147-155... 

In recent years studies have been developed in spontaneous disease models, gene knock out models and transgenic animals. These models provide information on the pharmacological action, pharmaco-kinetics and tolerability of a biotech products. 

Fig. 16.1 Both teratocarcinoma cells and normal embryonal stem cells behave alike. Both types of cells are accepted and incorporated into the inner layer of the blastocyst, when they are injected into the blastocyst cavity. They behave like normal embryonic stem cells, and the progeny of these cells are found in practically every cell and tissue of the chimeric mouse, where they differentiate normally. Moreover, they even form normal germ cells. Thus, teratocarcinoma stem cells, when separated from their undifferentiated cancerous daughter cells and transplanted into a compatible host, not only survive, they also do no harm to the host and do not make him cancerous. Incidentally, the capability of embryonic stem cells to be accepted by a recipient blastocyst is the basis of producing gene knock-out, chimeric mice, where a normal gene is replaced by an altered, engineered version of the gene. These chimeric mice then carry the mutation in their stem cells and transmit them to differentiated cells, where they are expressed. (With permission of Taylor and Francis, Inc. See Rg. 21-32 in ref. 1.)...

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