Genetic Manipulation Flashcards
Three Essential Features of a
Plasmid Cloning Vector
- Origin of replication
essential for self-
replication in host cells - Dominant selectable
marker gene
usually confers drug
resistance - One or more unique
restriction sites
Positive / Negative Selection
Positive Selection: Cells that have taken
up the recombinant plasmid will grow in
the presence of something, while those
that have not, die.
e.g. Antibiotic resistance
Negative Selection: Cells must lose
something to be identified, or survive.
e.g. loss of expression of a toxin gene, or
loss of b-galactosidase activity
Alpha Complementation:
Detecting recombinant plasmids
The E. coli lacZ gene encodes b-
galactosidase (b-gal).
b-gal converts the colourless substrate
X-Gal into a blue product.
Plasmid contains gene sequence for a-
peptide of b-gal.
E. coli cells lack this sequence in their
genome, lacZ-
Cells with b-gal activity produce blue
colonies when grown on X-Gal; cells
lacking b-gal produce white colonies.
Therefore, plasmids with foreign DNA
inserted in the a-peptide b-gal gene will
prevent active b-gal complementation
resulting in white colonies
Biofortification
Nutritional enhancement
Major goal is to address micronutrient
malnutrition
The GM approach offers some solutions
Golden Rice
vitamin A deficiency leading to 500,000 cases of
child blindness each year
Improved vitamin A nutrition could prevent
>600,000 child deaths per year
Engineer rice to contain b-carotene (provitamin
A), which gives it a yellow colour
Golden Rice 2 contains the phytoene synthase
gene from maize in an indica rice cultivar that is
consumed by 90% of the Asian population
B-Carotene Pathway
Recombinant Insulin
Until recombinant DNA technology, these
individuals had to purchase expensive
combinations from a variety of other animals
Now insulin is produced in E. coli in large vats
on a commercial scale
Patients can now obtain “Humulin”, a genetically
engineered human insulin
Use molecular biology to ‘improve’ insulin, e.g.
longer half-life in circulatory system
Other medicinal recombinant
proteins
Factor VIII – promotes blood clotting in treatment
of haemophilia
Tissue plasminogen activator (TPA) – dissolves
blood clots in treatment of heart attacks
Renin inhibitor – lowers blood pressure
Interleukin 2 – used to treat kidney cancer
Human growth hormone is used to treat pituitary
dwarfism in children
Used to be derived from human cadavers which can
transmit viral infections of central nervous system
Recombinant Erythropoietin
Erythropoietin (EPO) – stimulates red blood cell
production in situations of anaemia
EPO is a cytokine (protein signalling molecule)
EPO is a glycoprotein (carbohydrates attached to
certain amino acids).
As bacteria do not perform glycosylation, need to
express recombinant EPO in cultured human cells or
P. pastoris (yeast)
Obtain correct glycosylation pattern
Artificial Selection
- artificial selection: breeders choose which
organism to mate to produce offspring with
desired traits.
* They cannot control what genes are passed.
* When they get offspring with the desired traits,
the maintain them.
Three types of artificial selection:
A. selective breeding
B. hybridization
C. inbreeding
Selective Breeding
when animals with
desired characteristics are mated to
produce offspring with those desired traits.
* Passing of important genes to next
generation.
* Example: Champion race horses, cows
with tender meat, large juicy oranges on a
tree.
* Selective breeding occurs when you
choose the best male and female to
breed.
* This allows you to fine tune and control
the traits
* The offspring or babies will then have the
best traits.
* Then you continue to breed those
organism with the best traits, those traits
will be maintained.
Hybridisations
two individuals with unlike
characteristics are crossed to produce the best in both
organisms.
* Example: Luther Burbank created a disease resistant
potato called the Burbank potato.
* He crossed a disease resistant plant with one that had
a large food producing capacity.
* Result: disease resistant plant that makes a lot of
potatoes.
Inbreeding
breeding of organism that
genetically similar to maintain desired
traits.
* Dogs breeds are kept pure this way.
* Risk: since both have the same genes,
the chance that a baby will get a recessive
genetic disorder is high.
* Risks: blindness, joint deformities.
Cloning
Cloning: creating an organism that is an
exact genetic copy of another.
* identical twins are naturally created
clones.
How is cloning done
- Eggs are haploid
- Haploid: half the
chromosomes, 23 in
humans - Body cells are diploid:
- Diploid: two sets of
chromosomes, one from
mom and one set from
dad 46 in humans.
How could you clone
a human? - Step 1: An egg is
removed from a female
human - Eggs are haploid: 23
chromosomes. - The nucleus of the egg
is removed and is
thrown away.
23 - Step 2: A body cell is
removed from another
person. - The nucleus of the
body cell is removed - Body cells are diploid:
46 chromosomes.
46
Body Cell - Step 3:
- The nucleus of the
diploid body cell is put
into the egg. - This egg no longer
needs to be fertilized
since it has all 46
chromosomes.
46 - Step 4: The egg is then charged with
electricity to start mitosis. - Step 5: Its then put into a surrogate mother
so it can grow. - Its going to be genetically identical to the
parent of the body cell. - But it will be a baby.
- Plants and animals can be cloned.
