Cell Biology

1. CONSTRUCTION, ITS USES AND TYPES

2.  Also called genetic modification / genetic
manipulation / rDNA technology
 Direct manipulation of an organism’s DNA using
biotechnology
 Set of technologies used to change genetic makeup of
cells, including transfer of genes within and across
species boundaries to produce improved or novel
organisms
 New DNA obtained by isolating & copying gene of
interest (GI) using rDNA methods or by artificially
synthesising DNA

3. STEPS IN PLANT GENETIC
ENGINEERING

5.  The chosen piece of DNA is ‘cut’ from the source
organism using restriction enzymes.
 The piece of DNA is ‘pasted’ into a vector and the ends
of the DNA are joined with the vector DNA by ligation.
 The vector is introduced into a host cell, by a process
called transformation. The host cells copy the vector
DNA along with their own DNA, creating multiple
copies of the inserted DNA.
 The vector DNA is isolated from the host cells’ DNA
and purified.

7.  Nucleic acid molecules capable of autonomous
replication, can carry a gene and deliver it into a cell
 Different types of vectors includes :
 Plasmids
 Bacteriophages
 Cosmids
 Phagemids
 Artificial Chromosomes (BAC, YAC, HAC, MAC, Plant
derived AC)

8.  Synthetic chromosomes consisting of fragments of
DNA integrated into a host chromosome- introduced
into host cells to propagate and used to transfect
other cells, introducing new DNA.
 Useful in cloning larger fragments of DNA, as plasmids
can only contain up to 10,000 base pairs and phages
are hard to work with.
 Contain 300,000(BAC) to 1,000,000(YAC) base pairs-
reduces amount of runs needed for a large fragment
to be analyzed-easier and quicker to clone and
transform genes

9.  Linear or Circular
 Different types
 Bacterial Artificial Chromosome (BAC)
 Yeast Artificial Chromosome (YAC)
 P1 derived Artificial Chromosome (PAC)
 Mammalian Artificial Chromosome (MAC)
 Human Artificial Chromosome (HAC)
YAC – Cloning in yeast
BAC & PAC – Bacteria
MAC & HAC – Mammalian & Human cells.

10.  Developed by Mel Simmons and coworkers in 1992
 1st BAC vector – PBAC108L
 Plasmids constructed with replication origin of E.coli F
factor, so can be maintained in a single copy per cell
 Can hold DNA fragments of 75 to 300 kb
 Recombinant BACs introduced into E.coli by
electroporation
 Then rBAC replicates like an F factor

11.  Components :
 ori : origin of replication
 repE : for plasmid replication
and regulation of copy number
 parA and parB : for partitioning
F plasmids to daughter cells
during division and ensures
stable maintenance of the BAC
 Selectable marker for antibiotic
resistance. Some BACs also
have lacZ at cloning site
for blue/white selection
 T7 & Sp6 phage promoters for
transcription of inserted genes.

13.  Stable inserts and do not delete sequences
 Easy to manipulate
 Transforming efficiency higher than that of YAC
 Speedy growth of E.coli host
 Simpler to purify
 More user friendly
 Helpful in development of vaccines

14. • Fragment DNA contains unrelated genes may lead to
indirect, non-specific gene expression and
unanticipated changes in the cell phenotype.
• Generation and screening of recombinant BAC
constructs can be time-consuming and labor-intensive.
• Oversized BAC DNA constructs are more easily sheared
and degraded during manipulation before
transfection.
• Random recombination events may occur.
• Intra molecular rearrangements - reduce the
recombination efficiency and increase the rate of false
positive clones in some selection/counter-selection
approaches

15.  The P1 derived artificial chromosome (PAC) are DNA
constructs that are derived from the DNA of P1
bacteriophage. They can carry large amounts (about
100-300 kb) 0f other sequences for a variety of
bioengineering purposes.
 Host cell – E.coli

16.  Devised and first reported in 1987 by David Burk and
Olson
 pYAC3- first YAC developed.
 Special linear DNA vectors that resemble normal yeast
chromosome
 Circular double stranded DNA - contains a replication
origin (colE 1) compatible with E. coli in addition to
yeast replication origin or an yeast ARS element-useful
for amplification of the vector in E. coli.
 Clone large DNA segment (100 – 1000kb)

17.  By inserting large fragments of DNA, inserted
sequences can be cloned and physically mapped using
a process called chromosome walking.
 Initially used for the Human Genome Project- due to
stability issues abandoned for the use of Bacterial
Artificial Chromosome.

18.  Primary components -
autonomously replicating
sequence (ARS),
centromere (CEN) and
telomeres (TEL) from S.
cerevisiae.
 Selectable marker genes,
such as antibiotic
resistance and a visible
marker - select
transformed yeast cells.

19.  A YAC is built using an initial circular DNA plasmid,
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.

21.  Generating whole DNA libraries of the genomes of
higher organisms
 YAC clones used as hybridization probes for the
screening of cDNA libraries
 Study of regulation of gene expression by cis-acting,
regulatory DNA elements after the transfer of these
YACs from yeast to mammalian cells
 Used for isolation of functionally analogous
mammalian DNA sequences in order to develop MACs
Main difficulty in constructing MAC using YAC is isolation and
maintenance of mammalian centromere due to its large size and high
instability in yeast cell

22.  Very large DNA molecules are very fragile and prone to
breakage, leading to problem of rearrangement.
 High rate of loss of the entire YAC during mitotic
growth.
 Difficult to separate YAC from the other host
chromosomes because of their similar size.
 Separation requires sophisticated pulse-field gel
electrophoresis (PFGE).
 Yield of DNA is not high when YAC is isolated from
yeast cells.
 Clones tend to be unstable, with their foreign DNA
inserts often being deleted.

23.  Mammalian artificial chromosomes (MACs) are
conceptually similar to YACs, but instead of yeast
sequences they contain mammalian or human ones
 Like YACs rely on the presence of centromeric and
telomeric sequences and origin of DNA replication
 Involve autonomous replication and segregation in
mammalian cells, as opposed to random integration
into chromosomes (as for other vectors)
 Can be modified for their use as expression systems of
large genes, including not only the coding region but
can contain control elements

24.  Two principal procedures exist for the generation of
MACs.
1) In one method, telomere-directed fragmentation of
natural chromosomes is used. For example, a human
artificial chromosome (HAC) has been derived from
chromosome 21 using this method.
2) Another method involves de novo assembly of cloned
centromeric, telomeric, and replication origins in vitro .

25.  MAC vectors are difficult to assemble as compared to
YAC vectors
 Mammalian DNA has higher degree of repetition and
larger centromere and telomere regions
 Also the sequences necessary for chromosome
replication in mammalian system are not well defined
till now
 MAC vectors have application in the field of gene
therapy and eukaryotic protein expression and
production.

26.  Developed by Harrington et al., in 1997
 synthetically produced vector DNA, possessing
characters of human chromosome
 micro chromosome that can act as a new chromosome
in a population of human cells
 instead of 46 chromosomes, the cell could have 47
with 47th being very small
 Size 1/10th to 1/5th of human chromosome -6 – 10
megabases (Mb)

27.  able to carry new genes introduced by human
researchers
 researchers could integrate different genes that
perform a variety of functions, including disease
defence
 Using its own self-replicating and segregating systems,
HAC can behave as a stable chromosome that is
independent from the chromosomes of host cells
 Mitotically stable up to 6 months

28. COMPONENTS:
 Replication origin from which DNA duplication begins
 Centromere – functions in proper chromosome
segregation during cell division
 Telomere – protects ends of linear chromosomes

29. 2 approaches:
 Top-down method (telomere-associated chromosome
fragmentation)
 Bottom-up approach (de novo)

30.  Human chromosome transferred to chicken DT40 cells.
 Targeting linearized vectors containing telomeric
repeats, a selectable marker and sequences
homologous to q and p arms of chromosome
transected to chicken cells.
 Recombinational interaction between targeting
vectors and targeted sites at chromosome - forms new
telomeres - truncation of distal chromosomal arms.
 HAC generated by subsequent truncation of p-and q-
arms of chromosome.
 Usually a gene-loading loxP cassette is included into
HAC during chromosome truncation (i.e., included into
one of the targeting vectors).

31.  Final HAC vector transferred into Chinese hamster
ovary (CHO) cells. In CHO cells, a desired gene loaded
into loxP site of HAC by Cre/loxP-mediated
recombination
 From CHO cells, HAC with a gene of interest
transferred to desired recipient cells via a microcell-
mediated transfer technique.

33.  In de novo artificial chromosome by a bottom down
approach, exogenous chromosomes can be circular or
linear, created de novo from cloned chromosomal
components, either naturally occurring or synthetic
high – order α-satellite DNA arrays introduced on BAC,
YAC, or P1 artificial chromosome vectors, which have a
functional centromere and autonomously replicate
and segregate.

34.  Useful in expression studies as gene transfer vectors,
as a tool for elucidating human chromosome function
 Used to create transgenic animals to use as animal
models of human disease and for production of
therapeutic products
 Can carry genes to be introduced into the cells in gene
therapy
 Can carry human genes that are too long

35. Type Transgene
capacity
Host cell Advantages Disadvantages
BAC/PAC Up to 300 kb E.Coli  Can be infectiously
delivered
 Ease of purification
and modification .
 Medium transgene
capacity.
 May be
immunogenic
 Difficult to
chemically
transduce cells
YAC Up to 2.5 mb Yeast  Large transgene
capacity
 Ease to production
and modification
 Fragile construct
 Low transduction
efficiency
HAC Unlimited Cultured
human
cells
 Large transgene
capacity
 Naturally segregates
in human cells
 Non-immunogenic
 Non-integrating
 Fragile construct
 Difficult to purify
 Low transduction
efficiency

36.  PACs are chromosome-based vectors
(minichromosomes) for genetic engineering
Mini-chromosome
• Vector for plant artificial chromosome.
• Properties
1. No genes of their own, can be used as a super vector
to express foreign gene, minimum interference with
the host growth and development.
2. Stable during mitosis and meiosis, allow the foreign
gene to be faithfully expressed and transmitted.
3. Allow addition deletion and replacement of genes
with recent – genome editing technology.

37. Requirements for plant artificial
chromosome(mini chromosome)
1. Centromere
2. Telomere
3. Sufficient chromatin
4. Selectable marker transgene
5. Site specific recombination system

38. • Two methods
1. Essential components are assembly the gene outside
the plant cell, then transfer them into a plant cell.
But it creates more complexity to use.

39. 2. Mini chromosome constructed by telomere-mediated
chromosomal truncation(TMCT).
• Transformation of telomere –containing sequences
into genome, new telomeres at the site of integration.
• It must have functional centromere to keep stable
during cell division.

40.  The selectable marker gene(SMG) and the gene of
interest (GOI) in particular mini chromosome were
removed and replaced by those on donor plasmid
using specifically added restriction site on the mini
chromosome and homologous recombination.

41. • PACs after being generated in plants, cloned in-vitro
and transferred back into plants of the same species
they were generated in.
• Insertion is by protoplast fusion.
• To overcome the species specificity, allowing insertion
into alien species.

42.  Unlimited number of gene can be integrated and
expressed.
 Avoids linkage drag when transgene transferred to
other germplasm in breeding
 Prevents disruption of endogenous gene function.

43. TH
A
N
K
Y
O
U