Complete Genetic Engineering

1. 1
Lecture- 7

2.  1973 Plasmid
 1974 Bacteriophage lambda
 1977 Plasmid pBR322
Bacteriophage M13 (M13 mp1)
 1978 Cosmid
 1987 Yeast Artificial Chromosome-YAC
 1990 Bacteriophage P1
 1992 Bacterial Artificial Chromosome-
BAC
 1994 P1 Artificial Chromosome- PAC
 1997 human artificial chromosome-HAC
 2007 Maize Mini-Chromosomes
Cloning Vectors used in Genetic Engineering
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3. 3

4. Yeast Artificial Chromosomes
YACs are high insert capacity vectors
Contain yeast ARS, centromere and telomeres
But are associated with some problems:
i) 40 -60% of YACs from most libraries are chimeric
ii)40% of the YACs from most libraries are deleted
iii) Transformation efficiency is low and YACs are very
difficult to manipulate
4

5. Construction of Yeast
Artificial Chromosome
(YAC)
Cloning of large segments of
exogenous DNA into yeast by
means of artificial chromosome
vectors
250-400 kb
810 kb
1800 kb
Washington University, USA
Maynard Olson
Gruber Prize for Genetics 2007
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6. BACs are based on the naturally occurring F
plasmid of E. coli
Can be used to clone fragments of 300kbp or
longer
BACs have low copy no. origin of replication
and allow replication of clones at 1 copy/cell
BACs replicate clones faithfully across 60 -100
generations
Bacterial Artificial
Chromosomes
6

7. Construction of
Bacterial Artificial
Chromosome (BAC)
cloning and stable
maintenance of 300-kilobase-
pair of Human DNA in E. coli
using an F-factor-based vector
California Institute of
Technology
USA
Melvin Simon
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8.  Easy to manipulate
 Libraries are generated using bacterial hosts
with well defined properties
 Transformation efficiency is higher than that
obtained from YACs
 BACs are non chimeric
 BACs have very stable inserts and do not delete
sequences
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9.  PACs combine features of P1 vectors and BACs
 Have insert capacity in the range of 70 – 300kbp, and
average size of 150kbp
 PACs have low copy no. origin of replication based on P1
bacteriophage
 PACs allow replication of clones at one copy per cell and
replicate clones faithfully across 60 -100 generations
 PACs have a negative selection against non recombinants
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10. 1994
P.A. Ioannou and coworkers
developed
P1 artificial chromosome
(PAC)
cloning vector
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11. These are bacterial based systems that are easy to
manipulate
Libraries are generated using bacterial hosts with
well defined properties
Transformation efficiency is higher than that
obtained for YACs
PACs are non chimeric
PACs have very stable inserts and do not delete
sequences
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12. MACs are powerful tools for human
gene therapy and animal
transgenesis
Functional elements of such an
artificial chromosome are telomeres,
a centromere and a replication origin
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13.  Developed in 1997 – synthetic, self-
replicating
 ~1/10 size of normal chromosome
 Contains:
 ORI
 Centromere
 Telomeres
 Histones provided by host cell
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14.  Two approaches for construction of HACs :
 i) Top – down approach or TACF (telomere –
associated chromosome fragmentation)
Modifies natural chromosomes into smaller
defined minichromosomes in cultured cells
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15. ii) The bottom - up or assembly
approach
HACs are generated in human cells
by introducing defined
chromosomal sequences as naked
DNA including human telomeres and
genomic fragments containing
replication origins.
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16. Transfer and expression of genes in human
cells
De novo HACs are viable transfer vectors for
gene expression studies and can complement
genetic deficiencies in human cells
HACs contain regions of both euchromatin and
heterochromatin and therefore provide a
transcriptionally permissive environment for
gene expression
16

17. First shuttle
vector was
derived from
fusions from
E.coli vector
pBR322 and
the S.
aureus/B.
subtilis
plasmids pC194
and pUB110
Able to
replicate in E.
coli using one
origin and in
chosen host
using
alternative
replication
origin
The advantage
of shuttle
vector is that
E. coli can be
used as an
efficient
intermediate
for cloning
A shuttle vector is that vector which can
replicate in two hosts
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18. An expression vector is that vector in which
expression of the cloned gene can take place
Some specialized prokaryotic and eukaryotic
expression vectors have been constructed that
provide genetic elements for controlling
transcription, translation, protein stability and
secretion of the product of cloned gene from
the host cell
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19. Viral vectors for plants and
animals
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20.  These are modified in such a way so as to minimize the risk of
handling them
 They should have a minimal effect on the physiology of the cell it
infects
 The viral receptor on the host cell can be modified to target the
virus to a specific kind of cell
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21. For vector construction, following points should be
taken into consideration:
 The genome size should be small
 Vectors should have a broad host range
 Vector should be easy to transfer
 Vectors should be stable so that they can be
expressed in the plant
 There should not be any loss of infectivity of
viruses
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22. i)DNA viruses as vectors
ii)RNA viruses as vectors
DNA viruses
 Cauliflower Mosaic Virus
 Gemini virus
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23.  Animal viruses are used as vectors for transfer of
genes to cultured animal cells and living animals
 Viral vectors are used for gene expression in
cultrued animal cells and living animals
 Various classes of viral vectors:
retrovirus
adenovirus
herpesvirus
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24.  The type of cells or animals to be infected
 Whether infection leads to cell
transformation or lysis is required
 The number of genes to be to be expressed
 Whether virus infectivity is to be retained or
not
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25.  Retroviruses are a class of enveloped viruses
containing two copies of single strand sense(+) RNA
genome that replicates via ds DNA intermediate
 Viral genome is 7 – 10 kb in size, containing at least
three genes: gag, pol, env
 At each end of the genome are long terminal
repeats which include promoter/enhancer regions
and sequences involved in integration
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26.  P1 phage multiplies like a plasmid in the host
cell
 The genome of P1 phage is circular ds DNA of
100 kbp size which undergoes bidirectional
replication
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