Gerlai R, Williams S P, Cairns B, Van Bruggen N, Moran P, Shih A, Caras I, Sauer H, Phillips H S, Winslow J W
Neuroscience Department, Genentech, Inc., South San Francisco, CA 94080-4990, USA.
Exp Brain Res. 1998 Nov;123(1-2):24-35. doi: 10.1007/s002210050541.
Gene targeting using homologous recombination in embryonic stem (ES) cells offers unprecedented precision with which one may manipulate single genes and investigate the in vivo effects of defined mutations in the mouse. Geneticists argue that this technique abrogates the lack of highly specific pharmacological tools in the study of brain function and behavior. However, by now it has become clear that gene targeting has some limitations too. One problem is spatial and temporal specificity of the generated mutation, which may appear in multiple brain regions or even in other organs and may also be present throughout development, giving rise to complex, secondary phenotypical alterations. This may be a disadvantage in the functional analysis of a number of genes associated with learning and memory processes. For example, several proteins, including neurotrophins--cell-adhesion molecules--and protein kinases, that play a significant developmental role have recently been suggested to be also involved in neural and behavioral plasticity. Knocking out genes of such proteins may lead to developmental alterations or even embryonic lethality in the mouse, making it difficult to study their function in neural plasticity, learning, and memory. Therefore, alternative strategies to gene targeting may be needed. Here, we suggest a potentially useful in vivo strategy based on systemic application of immunoadhesins, genetically engineered fusion proteins possessing the Fc portion of the human IgG molecule and, for example, a binding domain of a receptor of interest. These proteins are stable in vivo and exhibit high binding specificity and affinity for the endogenous ligand of the receptor, but lack the ability to signal. Thus, if delivered to the brain, immunoadhesins may specifically block signalling of the receptor of interest. Using osmotic minipumps, the protein can be infused in a localized region of the brain for a specified period of time (days or weeks). Thus, the location and timing of delivery are controlled. Here, we present methodological details of this novel approach and argue that infusion of immunoadhesins will be useful for studying the role particular receptors play in behavioral and neural plasticity.
利用胚胎干细胞（ES细胞）中的同源重组进行基因打靶，提供了前所未有的精确性，借此人们可以操纵单个基因，并在小鼠体内研究特定突变的影响。遗传学家认为，这项技术消除了在脑功能和行为研究中缺乏高度特异性药理学工具的问题。然而，到目前为止已经清楚，基因打靶也存在一些局限性。一个问题是所产生突变的空间和时间特异性，其可能出现在多个脑区甚至其他器官中，并且在整个发育过程中也可能存在，从而导致复杂的继发性表型改变。这在对许多与学习和记忆过程相关基因的功能分析中可能是一个缺点。例如，最近有人提出，包括神经营养因子、细胞粘附分子和蛋白激酶在内的几种在发育中起重要作用的蛋白质，也参与神经和行为可塑性。敲除这类蛋白质的基因可能导致小鼠发育改变甚至胚胎致死，从而难以研究它们在神经可塑性、学习和记忆中的功能。因此，可能需要基因打靶的替代策略。在此，我们提出一种基于免疫粘附素全身应用的潜在有用的体内策略，免疫粘附素是一种基因工程融合蛋白，具有人IgG分子的Fc部分以及例如感兴趣受体的结合域。这些蛋白质在体内稳定，对受体的内源性配体表现出高结合特异性和亲和力，但缺乏信号传导能力。因此，如果将免疫粘附素递送至脑内，它们可能特异性阻断感兴趣受体的信号传导。使用渗透微型泵，可以在脑的局部区域在特定时间段（数天或数周）内注入该蛋白质。因此，递送的位置和时间是可控的。在此，我们展示了这种新方法的方法学细节，并认为注入免疫粘附素将有助于研究特定受体在行为和神经可塑性中所起的作用。
