Human Artificial Chromosomes
Mammalian Synthetic Biology
We seek to engineer human cells to remember past events and perform specific functions as a result. These systems are both transcriptional and post-transcriptionally driven. This includes cells that respond to drugs, disease states and the environment. We also develop novel ways to target cells with potentially therapeutic proteins with an emphasis on cells of the immune system and cancer. We develop computer simulations to model these systems.
Human Artificial Chromosomes
Since the 1990s, researchers have strived to generate human artificial chromosomes (HAC) to address the limitations of viral-based mammalian vectors. HACs are anticipated to allow large cloning capacities (megabase-scale), copy number control, long-term gene expression, preclusion of anti-viral responses, and reduced mutagenesis and insertion into host chromosomes. These efforts have consistently relied on excising and pasting centromeric fragments from natural human chromosomes into bacterial, phage and yeast artificial chromosomes (BACs, PACs and YACs). Yet, bacterial sequences can lead to increased mitotic instability, DNA rearrangement and concatenation in HACs. Though the two most salient HACs today can persist in cell lines for months and be manipulated in Chinese hamster ovary (CHO) cells using Cre-LoxP recombination, they have several disadvantages. The HACs are too large to manipulate in bacteria or yeast. Cloning and moving the HACs via microcell-mediated chromosome transfer (MMCT) takes months and requires an expertise rarely found outside labs that invented the HACs. Additionally, existing HACs lack precise design and often consist of megabases of random rearrangements. Genetic elements for chromatin modifications and insulation are critical to maintaining gene expression regulation. However, these remain to be integrated into existing HACs, possibly due to the prohibitive level of resources required. There are no HACs in distribution that can compare to BACs and YACs in terms of ease-of-use and design precision. In contrast, YACs containing well-defined basic functional units have been available since the 1980s, and are convenient to manipulate using conventional cloning protocols. YACs have been used by hundreds of studies and different researchers, extrapolating its utility so far as to study human genetic elements in YAC transgenic mice, and to perform multiplex assembly of exogenous biosynthetic pathways to produce diverse compounds in yeast.
Generating a HAC with simple methods of manipulation and transfection holds the same potential of transforming current practices and widening the breadth of possible experiments and synthetic biology designs in mammalian cell lines.
We have designed a novel minimal chromosome with an artificial centromere, telomeres, and insulator-separated gene expression domains based on current state of the art. We will construct different conformations (linear versus circular) and sizes (50 Kb to a few Mb) of our HAC to evaluate these hypotheses in the context of our approach, and ultimately derive a stable and user-friendly nucleomics tool for distribution. Over a few months, we will evaluate our HAC in terms of stability, segregation and maintenance of chromatin boundaries in various cell types such as human embryonic stem cells (hESCs)/induced pluripotent stem cells (iPSC) and commonly used cell lines HEK293, U2OS, HT1080, HeLa and CHO cells, using standard techniques to determine optimal HAC size and composition. This will be a well-defined and effective tool with many potential applications in cell line diversification/generation, animal transgenesis, and human gene therapy. We will apply the HAC in tissues-on-a-chip developed here at the Wyss Institute that hold tremendous potential for drug screening in primary cells functioning in artificial organs. For instance, the HAC can introduce genetic constructs and pathways to test their effect on a certain disease or drug treatment.
Efficient size-independent chromosome delivery from yeast to cultured cell lines. Brown DM, Chan YA, Desai PJ, Grzesik P, Oldfield LM, Vashee S, Way JC, Silver PA, Glass JI. Nucleic Acids Research. Link
The Genome Project-Write. Boeke JD et al. Science. PMID: 27256881
Researchers: Alina Chan (postdoctoral fellow)