ITBA-National Research Council, Via Ampere 56, Milano, Italy.
Cytotechnology. 1996 Jan;21(3):253-61. doi: 10.1007/BF00365348.
A mammalian artificial chromosome (MAC) may be assembled through the juxtapposition of three kinds of DNA elements: a centromere, several DNA replication origins, and two telomeric repeats. The resulting structure should be able to carry and express one or more selected genes (transgenes), introduced for specific purposes. The minimal length is unknown, but may be of several Mb.Of its basic elements, the telomeres may present lesser problems, in view of their simple composition and organization. Centromeres could be an issue, given their many unknowns. Mammalian DNA replication origins are at present poorly characterized, but it is expected that at least one may be contained within the MAC components, especially the transgene. Their overall assembly may require a combination of in vivo and in vitro approaches.A promising strategy aims at constructing two telomeric arms of a MAC, one of which may include the transgene. The two novel arms could acquire a functional centromere through recombination with the two arms of a resident chromosome. Alternatively, if the two telomeric constructs are also endowed with properly placed and oriented centromeric sequences, a centromere may be rescued in vivo by homologous recombination with the external parts of the centromere of the resident chromosome. Positive selection for the artificial arms and counterselection against the resident arms should facilitate the assembly process.The assembly of such construct would not change the ploidy number of the host cell. After loading of a transgene, however, the resulting MAC may be isolated and transferred into an expression cell, where it may represent a novel chromosomal element. In this case untoward effects to the host cell may derive from an ensuing dosage effect for the transgene(s) rather than from the presence of a MAC per se.A MAC may contribute to a deeper understanding of the structural requirements for chromosomal function and evolution as well as the mechanism of chromatin formation. It should also help in the development of second generation vectors for transfer of Mb-long DNA sequences, as required for properly regulated mammalian gene function as well as, possibly, for therapy.
哺乳动物人工染色体 (MAC) 可以通过三种 DNA 元件的并置组装而成：着丝粒、几个 DNA 复制起点和两个端粒重复序列。由此产生的结构应该能够携带和表达一个或多个选定的基因（转基因），这些基因是为特定目的而引入的。最小长度未知，但可能为数 Mb。
在其基本元件中，端粒的组成和组织简单，因此可能不会出现太大问题。着丝粒则可能是一个问题，因为它们有很多未知因素。哺乳动物 DNA 复制起点目前的特征描述较差，但预计至少有一个可能包含在 MAC 成分中，特别是在转基因中。它们的整体组装可能需要体内和体外方法的结合。
一种有前途的策略旨在构建 MAC 的两个端粒臂，其中一个臂可能包含转基因。这两个新臂可以通过与常驻染色体的两个臂重组获得功能性着丝粒。或者，如果这两个端粒构建体也具有适当放置和定向的着丝粒序列，则通过与常驻染色体的着丝粒外部部分进行同源重组，体内可能会拯救着丝粒。对人工臂的正选择和对常驻臂的反选择应有助于组装过程。
这种构建体的组装不会改变宿主细胞的倍性。然而，在加载转基因后，所得的 MAC 可以被分离并转移到表达细胞中，在那里它可能代表一个新的染色体元件。在这种情况下，对宿主细胞的不良影响可能源自转基因（多个）的剂量效应，而不是 MAC 本身的存在。
MAC 可能有助于更深入地了解染色体功能和进化的结构要求以及染色质形成的机制。它还应该有助于开发第二代载体，用于转移 Mb 长的 DNA 序列，这是适当调节哺乳动物基因功能所必需的，可能还需要用于治疗。
