A human artificial chromosome (HAC) is a
microchromosome that can act as a new
chromosome
A chromosome is a package of DNA containing part or all of the genetic material of an organism. In most chromosomes, the very long thin DNA fibers are coated with nucleosome-forming packaging proteins; in eukaryotic cells, the most import ...
in a population of
human
Humans (''Homo sapiens'') or modern humans are the most common and widespread species of primate, and the last surviving species of the genus ''Homo''. They are Hominidae, great apes characterized by their Prehistory of nakedness and clothing ...
cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10
megabases (Mb) in size instead of 50–250Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, includin
disease defense
Alternative methods of creating
transgene
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...
s, such as utilizing
yeast artificial chromosomes and
bacterial artificial chromosome
A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partiti ...
s, lead to unpredictable problems. The genetic material introduced by these
vector
Vector most often refers to:
* Euclidean vector, a quantity with a magnitude and a direction
* Disease vector, an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematics a ...
s not only leads to different expression levels, but the inserts also disrupt the original genome. HACs differ in this regard, as they are entirely separate chromosomes. This separation from existing genetic material assumes that no
insertional mutants would arise.
This stability and accuracy makes HACs preferable to other methods such as
viral vector
A viral vector is a modified virus designed to gene delivery, deliver genetic material into cell (biology), cells. This process can be performed inside an organism or in cell culture. Viral vectors have widespread applications in basic research, ...
s, YACs, and BACs. HACs allow for delivery of more DNA (including
promoters and
copy-number variation
Copy number variation (CNV) is a phenomenon in which sections of the genome are repeated and the number of repeats in the genome varies between individuals. Copy number variation is a type of structural variation: specifically, it is a type of ...
) than is possible with viral vectors.
Yeast artificial chromosomes and
bacterial artificial chromosome
A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually '' E. coli''. F-plasmids play a crucial role because they contain partiti ...
s were created before human artificial chromosomes, which were first developed in
1997
Events January
* January 1 – The Emergency Alert System is introduced in the United States.
* January 11 – Turkey threatens Cyprus on account of a deal to buy Russian S-300 missiles, prompting the Cypriot Missile Crisis.
* January 1 ...
. HACs are useful in expression studies as
gene transfer vector
Vector most often refers to:
* Euclidean vector, a quantity with a magnitude and a direction
* Disease vector, an agent that carries and transmits an infectious pathogen into another living organism
Vector may also refer to:
Mathematics a ...
s, as a tool for elucidating human chromosome function, and as a method for actively annotating the
human genome
The human genome is a complete set of nucleic acid sequences for humans, encoded as the DNA within each of the 23 distinct chromosomes in the cell nucleus. A small DNA molecule is found within individual Mitochondrial DNA, mitochondria. These ar ...
.
History
HACs were first constructed ''de novo'' in 1997 by adding alpha-satellite DNA to telomeric and genomic DNA in human
HT1080
HT1080 is a fibrosarcoma cell line which has been used extensively in biomedical research. The cell line was created from tissue taken in a biopsy of a fibrosarcoma present in a 35-year-old human male. The patient who supplied the sample had not u ...
cells. This resulted in an entirely new
microchromosome that contained DNA of interest, as well as elements allowing it to be structurally and mitotically stable, such as telomeric and centromeric sequences.
Due to the difficulty of ''de novo'' HAC formation, this method has largely been abandoned.
Construction methods
There are currently two accepted models for the creation of human artificial chromosome vectors. The first is to create a small
minichromosome by altering a natural human chromosome. This is accomplished by truncating the natural chromosome, followed by the introduction of unique genetic material via the
Cre-Lox system of recombination. The second method involves the literal creation of a novel chromosome ''de novo''. Progress regarding ''de novo'' HAC formation has been limited, as many large genomic fragments will not successfully integrate into ''de novo'' vectors.
Another factor limiting ''de novo'' vector formation is limited knowledge of what elements are required for construction, specifically
centromeric sequences.
Challenges involving centromeric sequences are being overcome.
Applications
A 2009 study has shown additional benefits of HACs, namely their ability to stably contain extremely large genomic fragments. Researchers incorporated the 2.4Mb dystrophin gene, in which a mutation is a key causal element of
Duchenne muscular dystrophy
Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy predominantly affecting boys. The onset of muscle weakness typically begins around age four, with rapid progression. Initially, muscle loss occurs in the thighs and pe ...
. The resulting HAC was mitotically stable, and correctly expressed dystrophin in chimeric mice. Previous attempts at correctly expressing dystrophin have failed. Due to its large size, it has never before been successfully integrated into a vector.
In 2010, a refined human artificial chromosome called 21HAC was reported. 21HAC is based on a stripped copy of human chromosome 21, producing a chromosome 5Mb in length. Truncation of chromosome 21 resulted in a human artificial chromosome that was mitotically stable. 21HAC was also able to be transferred into cells from a variety of species (mice, chickens, humans). Using 21HAC, researchers were able to insert a herpes simplex virus thymidine kinase coding gene into tumor cells. This "suicide gene" is required to activate many antiviral medications. These targeted tumor cells were successfully, and selectively, terminated by the antiviral drug
ganciclovir in a population including healthy cells. This research opens a variety of opportunities for using HACs in gene therapy.
In 2011, researchers formed a human artificial chromosome by truncating chromosome 14. Genetic material was then introduced using the
Cre-Lox recombination system. This particular study focused on changes in expression levels by leaving portions of the existing genomic DNA. By leaving existing telomeric and sub-telomeric sequences, researchers were able to amplify expression levels of genes coding for erythropoietin production over 1000-fold. This work also has large gene therapy implications, as erythropoietin controls red blood cell formation.
HACs have been used to create
transgenic animals for use as animal models of human disease and for production of therapeutic products.
See also
*
Plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
*
Cosmid
*
Fosmid
References
{{Nucleic acids
Molecular biology