Genetic Engineering

Fatchul Anam Nurlaili

4
pollen
grain

ovule

HHrr
high yield
low resistance

hhRR
low yield
high resistance

The F1 consists of
plants with high yield
and good resistance

zygote

5
Can you see any disadvantages in this method of
manipulating genes ?

Try working out what would happen if you tried to breed from
the F1
Work out the various gene combinations in the gametes

Put them into a

4x4 Punnett Square

F1 cross

F1 cross HhRr x HhRr

Possible combination
of genes in gametes

HR

Hr

hR

HR

Hr

hR

hr

hr

HR

HHRR

HHRr

HhRR

HhRr

Hr

HHRr

HHrr

HhRr

Hhrr

hR

HhRR

HhRr

hhRR

hhRr

hr

HhRr

Hhrr

hhRr

hhrr

The F1 does not breed true. Of the 16 possible combinations

of genes, 7 do not have the combined beneficial genes

erbreeding transfers the complete genome of one variety to

other.

s means that many new and unpredictable gene combinatio

y be formed in addition to those intended

is method of genetic recombination can take place only betw

rieties of the same or closely related species

netic engineering makes it possible to transfer single gene

he genes can also be transferred from one species to a totall

fferent species

Genetic engineering

9
There are several ways in which genes from one organism can be
inserted into a different organism

They can be coated on to microscopic gold particles and fired

into the cells

They can be delivered by viruses

They can be transmitted by using structures, called plasmids,

present in bacteria

For example, the human gene for making insulin can be transferred
to bacteria, which are then allowed to reproduce in a culture medium
from which the insulin can be extracted

Plasmids

DNA is the genetic material of most

organisms (from bacteria to humans)
Plasmid

Chromosome: Most bacteria have one circular DNA chromosome ranging in

size from 1,000 to 8,000 kilobase pairs.
Plasmid: Extrachromosomal genetic element also made of a circular DNA
molecule.
Bacterial Genome: The collection of all of the genes present on the
bacterias chromosome or its extrachromosomal genetic elements.

10

A bacterium
in addition to a loop of DNA

bacteria also contain numerous

rings of DNA called plasmids

cell wall

cytoplasm

cell membrane

0.001mm

the plasmids can be

extracted and used for
genetic engineering

plasmid

11

Inserting a
gene
human DNA
strand
restriction
enzyme cuts
plasmid

the insulin gene

is inserted into
the plasmid

insulin
gene

the same
restriction
enzyme cuts
the insulin gene
out of the
human DNA

12
The recombinant plastids are
inserted into a bacterium *

Recombinant
plastids

the insulin gene makes the

bacterium produce insulin

13
Only about 1 in 100,000 bacteria take up the recombined plasmids
There are techniques for identifying and isolating these bacteria
The bacteria with the insulin gene are then allowed to reproduce
in a culture solution from which the insulin can be extracted*

Human growth hormone can be made in a similar way

Factor VIII, needed by haemophiliacs, (blood clotting disorders)
can be produced from hamster cells containing plasmids with the
factor VIII genes

Chymosin, used for clotting milk in cheese-making, can be

produced from yeast cells with recombinant plasmid DNA
Applications

14

As well as producing useful substances from genetically altered

cells, whole organisms can be genetically modified.
Some examples are .

A bacterial gene which makes an insecticide can be introduced into

crop plants, e.g. maize and cotton, to make them resistant to attack
by moth caterpillars

A gene which confers resistance to herbicides has been inserted

into crop plants so that spraying kills weeds but not the crop plants
A gene introduced to oilseed rape makes the oil more suitable
for commercial processes, e.g. detergent production

Genes which control the production of human enzymes have been

inserted into sheep so that the enzymes can be recovered from
their milk
Applications

15
Genetic engineering does not always have to involve gene transfer
between unrelated organisms

Genes in a single organism can be modified to improve their

characteristics or their products

A gene for the production of carotene (a precursor of Vitamin A)

has been introduced to rice to benefit countries where rice is the
staple diet and Vitamin A deficiencies are common*

The next slide shows tomatoes which have been genetically

modified to suppress production of an enzyme which causes the
fruit to soften as it ripens. This improves the keeping qualities

Applications

Cloning

18

en organisms reproduce asexually, all the offspring receive a

of genes from the parent.
As a result they are identical to each other and
to the parent
Examples are :
Bacteria and single-celled organisms
Plants with vegetative reproduction by bulbs, corms etc.
Fungi
Some of the lower invertebrates

population of identical individuals arising from asexual

eproduction is called a clone

19

A clone of crocuses

cells in sheep As
mammary gland

21
egg cell (ovum)
from sheep B

diploid
nucleus
one cell
isolated
the two cells
are fused together *

nucleus
removed

cell division produces

early embryo
embryo implanted
in uterus of sheep C

cloned lamb
born
Dolly

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Sheep, pigs, horses, cows and, by now, probably many more animals
have been cloned

So far, this is being done on an experimental basis

Hundreds of embryos have to be prepared and implanted to obtain

one or two successful births

If the process becomes cheap and reliable it means that beneficial

genes will be present in all the offspring, thus eliminating the
chances of their being lost during conventional breeding

Before the early embryo is implanted in the surrogate mother, it can

be broken up into its individual cells. Each of these can develop into
a new embryo

23

fertilised frog egg

at the 8-cell stage, any one of these
cells can develop into a frog
cell division to form
an embryo

growth and development to

produce tadpole and frog

24

Clone of
frogs

each cell can develop into a frog

8-cell frog embryo

cells separated

25

cells from the 8-cell embryo are called embryonic stem ce

because each one can form all the cells and tissues to
roduce a complete frog

After the 16-cell stage, the cells lose this ability

and can only
produce specialised cells such as blood, bone and
ells capable
of dividing to produce specialised cells are
nerve cells
alled stem cells

pecialised cells normally lose the power to divide and may ha

limited life span

he tissues produced by specialised cells usually contain som

tem cells which retain the power of division

29
section through a 10-day
human embryo

these cells will contribute

to the placenta

stem cells transferred

to culture dish

0.5 mm

nutrient medium*
these cells will form
the embryo (stem cells)

stem cells cultured

(cloned)
Human ESCs

30
All the cells in the body have a full set of genes

When the cells become specialised, they lose their ability to divide
and many of the genes are switched off

For example, the genes for producing hydrochloric acid in a stomach

cell would not be functional in a skin cell

Even though tissues consist mainly of specialised cells, most of them

also contain their own stem cells

It may become possible to treat stem cells from specialised tissues

with hormones and growth factors that cause them to produce a
wider range of specialised cells*

Applications of stem
cells

31

st applications of stem cells are in the experimental stage, ar

ergoing clinical trials or have been tried on very few patients
Possibilities are :

Replacement of damaged tissues such as heart muscle, sk

bone and cartilage

Treatment of disease, e.g. diabetes by injecting islet cells

into the pancreas; or Parkinsons disease by injecting nerv
stem cells into the brain

he stem cells can be derived from the patients own tissue,

ection by the immune system is avoided

What Does It Mean: To

Clone?
Clone: a collection of molecules or cells, all identical
to an original molecule or cell
To "clone a gene" is to make many copies of it - for
example, by replicating it in a culture of bacteria.
Cloned gene can be a normal copy of a gene (=
wild type).
Cloned gene can be an altered version of a gene (=
mutant).
Recombinant DNA technology makes manipulating
genes possible.

Restriction Enzymes
Bacteria have learned to "restrict" the
possibility of attack from foreign DNA by
means of "restriction enzymes.
Cut up foreign DNA that invades the cell.
Type II and III restriction enzymes cleave DNA
chains at selected sites.
Enzymes may recognize 4, 6 or more bases in
selecting sites for cleavage.
An enzyme that recognizes a 6-base sequence
is called a "six-base cutter.

Basics of type II Restriction

Enzymes
No ATP requirement.
Recognition sites in double stranded DNA have a
2-fold axis of symmetry a palindrome.
Cleavage can leave staggered or "sticky" ends or
can produce "blunt ends.

Type II restriction enzyme nomenclature

Why the funny names?

EcoRI Escherichia coli strain R, 1st enzyme

BamHI Bacillus amyloliquefaciens strain H, 1st
enzyme
DpnI Diplococcus pneumoniae, 1st enzyme
HindIII Haemophilus influenzae, strain D, 3rd
enzyme
BglII Bacillus globigii, 2nd enzyme
PstI Providencia stuartii 164, 1st enzyme
Sau3AI
Staphylococcus aureus strain 3A, 1st
enzyme
KpnI Klebsiella pneumoniae, 1st enzyme

DNA Ligase joins DNA fragments

together
Enzymes that cut with staggered cuts result in
complementary ends that can be ligated
together.
HindIII - leaves 5 overhangs (sticky)

5AAGCTT3

5AAGCTT3

3TTCGAA5

3TTCGAA5

Sticky ends that are complementary (from

digests with the same or different enzymes) can
be ligated together.
Sticky ends that are not complementary cannot

DNA Ligase can also join blunt ends

DNA fragments with blunt ends generated by different
enzymes can be ligated together (with lower
efficiency), but usually cannot be re-cut by either
original restriction enzyme.

SmaI CCCGGG
DraIAAATTT

CCCGGG
AAATTT
CCCTTT
AAAGGG

Ligations that re-constitute a SmaI or DraI site (CCCGGG or AAATTT)

can be re-cut by SmaI or DraI.
Mixed ligation products (CCCTTT + AAAGGG) cannot be re-cut by
SmaI or DraI.

Plasmids vehicles for

cloning

Plasmids are naturally occurring

extrachromosomal DNA
molecules.

Ampr

Ori

pBR322
4361bp

Plasmids are circular, doublestranded DNA.

Tetr

Plasmids are the means by which

antibiotic resistance is often
transferred from one bacteria to
another.

LacZ
MCS

Plasmids can be cleaved by

restriction enzymes, leaving
sticky or blunt ends.
Artificial plasmids can be
constructed by linking new DNA
fragments to the sticky ends of

pUC18
Ori
Ampr

Cloning Vectors
Older cloning vector

A cloning vector is a plasmid that

can be modified to carry new
genes.
Plasmids useful as cloning vectors
must have:
An origin of replication.
A selectable marker (antibiotic
resistance gene, such as ampr
and tetr).
Multiple cloning site (MCS) (site
where insertion of foreign DNA
will not disrupt replication or
inactivate essential markers).
Easy to purify away from host
DNA.

Ampr
Ori

pBR322
4361bp

Tetr

Newer cloning vector

LacZ
MCS

pUC18
Ampr

Ori

Chimeric Plasmids
Named for mythological beast
(chimera) with body parts from
several creatures.
After cleavage of a plasmid with a
restriction enzyme, a foreign DNA
fragment can be inserted.
Ends of the plasmid/fragment are
closed to form a "recombinant
plasmid.
Plasmid can replicate when placed
in a suitable bacterial host.

CF
TR
LacZ
MCS

pUC18-hCFTR
Ori
Ampr

DNA cloning requires

restriction endonuclease
and DNA ligase

Consider a plasmid with a unique EcoRI

site:
5'NNNNGAATTCNNNN3'
3NNNNCTTAAGNNNN5'
An EcoRI restriction fragment of foreign
DNA can be inserted into a plasmid
having an EcoRI cloning site by:
a) cutting the plasmid at this site with
EcoRI,
b) annealing the linearized plasmid with
the EcoRI foreign DNA fragment, and,
c) sealing the nicks with DNA ligase.
5'NNNNGAATTCNNNN3'
3'NNNNCTTAAGNNNN5