Why we are investigating artificial chromosome vectors.

Genome and chromosomes

All living organisms store genetic information to define their distinct properties and vital activities in the form of chromosomes, which are composed of genomic DNA and protein molecules. In eukaryotes, the packaging of genetic information in chromosomes is essential for proper gene expression during cell cycle progression and cell differentiation and for the accurate transmission of genomic DNA to progeny cells.

The human genome is composed of 3 billion base pairs of DNA, which is 1 m in length. Genomic DNA is distributed into 23 distinct chromosomes: 22 autosomes and one of two types of sex chromosomes (X or Y) in a haploid germ line cell. Diploid human cells (body cells) contain 46 chromosomes: two copies of each of the 22 autosomes and two sex chromosomes, either XX (female) or XY (male).

Analyses of the Human Genome Project revealed that the human genome contains about 22,000 genes. These genes occupy only 5% of the genome, and the remaining 95% is related to the function, behavior, and inheritance of the chromosomes. Further analyses are needed to better understand chromosomes as vehicles for genes.

What is a HAC?

A human artificial chromosome (HAC) is a mini-chromosome that is constructed artificially in human cells. Using its own self-replicating and segregating systems, a HAC can behave as a stable chromosome that is independent from the chromosomes of host cells.
The essential elements for chromosome maintenance and transmission are the following three regions:
(1) the greplication origin,h from which the duplication of DNA begins,
(2) the gcentromere,h which functions in proper chromosome segregation during cell division, and
(3) the gtelomere,h which protects the ends of linear chromosomes.

A study of functional regions of the chromosome was conducted first in budding yeast. A yeast artificial chromosome (YAC) with artificially constructed DNA encompassing the three regions mentioned above was generated in yeast cells. In prokaryotes, a bacterial artificial chromosome (BAC) was constructed on the basis of the naturally occurring F plasmid of E. coli.

Thus, by the study of functional regions of the chromosome, gartificial chromosome technologyh that reconstructed in vivo mechanisms of chromosome maintenance and transmission was established. Because very large genomic DNA fragments can be cloned into BACs and YACs, these artificial chromosomes have been used widely as vectors in basic genetic engineering technology, such as genome analysis and the production of transgenic animals, since the 1990s.

On the other hand, the particular DNA sequences specifying the replication origin and centromere were unresolved in higher eukaryotes such as mammals. The research group of Tsuneko Okazaki, Ph.D., the founder of Chromo Research, investigated these sequences and succeeded in building a HAC with type I alphoid DNA from human chromosome 21. HAC, which is maintained as an extra chromosome, duplicates synchronously with host cell chromosomes at each host cell division and is transmitted stably to daughter cells.

Production of HAC by bottom-up construction strategies

Tsuneko Okazaki and her collaborators discovered that HACs were frequently generated de novo in the human fibroblast cell line HT1080 upon introduction of precursor DNA constructs in YACs or BACs. These YACs or BACs contained up to 70 kilobases of humanƒ¿-satellite (alphoid) DNA derived from human chromosome 21, as well as marker genes and, in the case of YAC constructs, telomere sequences at both DNA ends.
A HAC can be detected by FISH analysis as a gmini-chromosomeh generated by multimerization of the introduced DNA. A HAC made from a YAC is linear with telomeric structures, whereas a HAC generated from a BAC is circular and lacks telomeres. Dr. Okazakifs findings revealed an important principal: gtype I alphoid DNA with frequent CENP-B boxes is necessary for de novo centromere/kinetochore formation.h On the basis of these results, Chromo Research scientists were the first to reproducibly manufacture HACs. We are applying for patents of these technologies in major countries, have already been issued patents in the USA, Australia, some European countries, and Japan, and are under investigation in Canada. Therefore, Chromo Research has global predominance in the technology.
The gbottom-up constructionh strategy of Chromo Research involves the de novo construction of HACs by introducing necessary DNA elements for the maintenance of chromosome function into cells. On the other hand, gtop-down constructionh refers to the truncation of natural chromosomes into smaller sizes by using targeting vectors containing telomeric sequences.

production of HAC

Construction of HAC vectors and various useful applications

In bottom-up construction, multimerization of transfected DNA occurs during HAC formation. Thus, HACs that contain transgenes are generated de novo from precursor BAC or YAC vectors that contain the transgene and an alphoid array in separate vectors.
We produced a HAC that carries a site-directed insertion system. Because a transgene (cDNA or genomic DNA) can be inserted at a certain position in this HAC, the transgene in the HAC can be expressed in mammalian cells in a promoter-dependent manner under the desired stable control.

We confirmed that microcell-mediated chromosome transfer (MMCT) enables HACs to be transferred into various cell types and maintained stably. At present, we have studied stable maintenance of HACs in the following cell lines: mouse embryonic stem (ES) cells, human mesenchymal stem cells, Chinese hamster ovary (CHO) cells, chicken B (DT40) cells, pig kidney (PK15) cells, and K562 cells etc.. HACs can be transferred into mouse embryonic stem cells by MMCT, so a transgenic mouse containing exogenous genes can be readily created. The establishment of a reliable method to create a transgenic animal will enable HAC vector utilization for gene therapy and regenerative medicine.


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