Human Artificial Chromosomes (HAC) are fascinating extrachromosomal molecules that stay independently from the host genome and are capable of segregating as efficiently as endogenous chromosomes. It has been proven that HAC are potential tools for both basic chromosome behavioural research and agents for gene therapy purposes. My DPhil project is divided into two main themes. The first theme was to develop a novel artificial chromosome in mouse embryonic stem cells. The second theme was to understand the factors affecting chromosome stability which may also affect the efficiency of artificial chromosome formation. so that our protocol for better HAC preparation can be refined. There are six results chapters in my thesis. The first three chapters described how I developed human artificial chromosomes in mouse embryonic stem cells. Initially, vectors containing a long stretch of human alphoid DNA were delivered to mouse cells using the Herpes Simplex Virus-I (HSV-l) amplicon system but the efficiency was low. Next, mouse pericentromeric and centromeric DNAs were employed for mouse artificial chromosome (MAC) via HSV-l system. However, the efficiency remained the same. Finally, I used the Microcell-Mediated Chromosome Transfer (MMCT) system to transfer HAC from HTl 080 cells into mouse ES cells and successfully established HAC in ES which were highly stable. The results obtained in this first part of my thesis suggested that to increase HAC formation efficiency it would be necessary to improve the techniques of HSV-I delivery and MMCT. Moreover, it would also be important to better characterize factors affecting chromosome behaviour. The last three results chapters focus on factors affecting chromosomes stability and improving the HSV -1 delivery system and MMCT. I undertook an in vivo study of whole cell fusion experiments with the aid of live cell irnaging system, and found that histone H2B proteins underwent a dynamic assembly/disassembly processes. Live cell imaging of MMCT suggested that the microcell delivery is a very slow process and the results may lead to a refinement of the MMCT protocol. I found it is possible to generate a single HAC using two HSV-l amplicons containing two different constructs, potentially doubling the HSV-l HAC capacity from 150 kb to 300 kb. The last chapter illustrated how the expression of non- coding centromeric satellites impaired chromosome stability in both human cultured and human embryonic stem cells. The findings revealed that non-coding centromeric RNA plays an important role on chromosome stability that might be important for artificial chromosome development.