artificial chromosome vector

1. Artificial chromosome
. vector
Presented by :
pushpendra singh gahlot
M.sc botany
University of kota
ag1805x

2. Artificial Chromosome Vectors
● Artificial chromosomes are DNA molecules or fragment
assembled in vitro from defined constituents, which
guarantee stable maintenance of large DNA fragments
with the properties of natural chromosomes.
● Artificial
sequencing
chromosomes are useful for genome
programmes, for functional characterization
of entire genomic regions and for the transduction of
nonhumanlarge DNA segments into human and
mammalian cells.
● Types: BAC, YAC, PAC, HAC

3. Bacterial artificial chromosome
● A bacterial artificial chromosome (BAC) is a DNA
construct, based on a functional fertility plasmid (or
cloning inF-plasmid), used for transforming and
bacteria, usually E. Coli.
● uptoThey are capable of carrying approximately
300 kbp of inserted DNA sequence.
Ex: pBAC108L
pBeloBAC11

4. Common gene Componentsof BAC
Ori s:origin of replication from Ecoli f
plasmid
Rep E: encode a rep protein
parA,parB,parC,: partitioning of F plasmid
during cell division
CM : chloramphenical resitance
Cos N :lambda phage cos site
lacZ: β galactosidase gene
T7:t7 bacterio phage driven promoter

5. Fig.: BacterialArtificial
Chromosome
pBAC108L

6. Applications of BAC
● Contribution to models of disease: Inherited disease
● geneticBACs are now being utilized in modeling
diseases, often alongside transgenic mice.
● BACs have been useful in this field as complex genes
may have several regulatory sequences upstream of
the encoding sequence, including various promoter
sequences that will govern a gene's expression level.
● BACs have been used to study neurological diseases such
as Alzheimer's disease or as in the case of aneuploidy
associated with Down syndrome. There have also been
instances when they have been used to study specific
oncogenes associated with cancers.

7. Applications of BAC [contd.]
● Contribution to models of disease: Infectious disease
● The genomes of several large DNA viruses and
RNA viruses have been cloned as BACs.
● These constructs are referred to as "infectious
clones".
● The infectious property of these BACs has made
the study of many viruses such as the
herpesviruses, poxviruses and coronaviruses
more accessible.

8. Applications of BAC [contd.]
● Sequencing: BACs are often used to sequence the
genome of in genome projects, for example
the Human
organisms
Genome Project. A short piece of the
organism's DNA is amplified as an insert in BACs, and
then sequenced. Finally, the sequenced parts are
rearranged in silico, resulting in the genomic sequence of
the organism.

9. Yeast artificial chromosome
●
●
Yeast artificial chromosomes (YACs) are genetically
engineered chromosomes derived from the DNA of the
yeast, Saccharomyces cerevisiae
Yeast artificial chromosome (YAC) vectors allow the
cloning, within yeast cells, of fragments of foreign
genomic DNA that can approach 500kbp in size.
The YAC was devised and first reported in 1987 by
David Burke

10. Construction of yac
 YAC is built using an initial circular
DNA plasmid, which is typically cut into a
linear DNA molecule using restriction
enzymes; DNA ligase is then used to ligate
a DNA sequence or gene of interest into
the linearized DNA, forming a single large,
circular piece of DNA.

12. Application of yac
 , YACs can be utilized to clone and
assemble the entire genomes of an
organism.
it can be used to find genes, segments of
DNA that code for a specific protein or
phenotype,

13. Limitations of using YAC vectors
prone toVery large DNA molecules are very fragile and
breakage, leading to problem of rearrangement.
Difficult to separate YAC from the other host chromosomes
because of their similar size.
Yield of DNA is not high when the YAC is isolated from
yeast cells.
Clones tend to be unstable, with their foreign DNA inserts
often being deleted.

14. P1 derieved artificial chromose
 P1 derived artificial chromosome are DNA
cunstruct that are derived from dna of p1
bacteriophage they can carry large
amount(about 100-300 kbs ) of other
sequence for a variety of bioengineering
purose.
 P1 was developed as a cloning vector
by Nat Sternberg and colleagues in the
1990s.

15. Human artificial chromosome
● chromosomeA human artificial
microchromosome that can act
(HAC) is a
as a new chromosome
in a population of human 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 for
natural chromosomes, and
instead
able
of 50-250 Mb
to carry new genes
introduced by human researchers.
● Ideally, researchers could integrate different genes that
perform a variety of functions, including disease
defense.

16. Construction methods HAC
● There are currently two accepted models for the
creation of human artificial chromosome vectors.
➔
➔

17. Construction methods HAC [contd.]
● In engineered chromosome by a top-down approach,
minichromosomes or chromosomes
chromosomes generated
derived
by
from
natural
of chromosomes, telomere-directed
radiation-induced
endogenous
fragmentation
chromosome
chromosome
breakage,
breakage,
or
containing an
functional centromere. The chromosomes
endogenous
can then be
microcell-mediatedtransferred into other cell lines by
chromosome transfer (MMCT).

18. ● In de novo artificial chromosome
chromosomes can be
by a bottom-up
circular or
novo from cloned
approach, exogenous
linear, created de
components, either naturally occurring
chromosomal
or synthetic
high-order α-satellite
bacterial artificial chromosome
DNA arrays introduced on
(BAC), yeast artificial
chromosome (YAC), or p1-derived artificial chromosome (PAC)
vectors, which have a functional centromere and
autonomously replicate and segregate.
Construction methods HAC [contd.]

19. Applications of HAC
● HACs are useful in expression studies as gene transfer
vectors, as a tool for elucidating human chromosome
function, and as a method for actively annotating the
human genome.
● HACs have been used to create transgenic animals for use
as animal models of human disease and for production of
therapeutic products.
● HAC can carry genes to be introduced into the cells in
gene therapy.

20. Thank You
ag1805x