Chapter 31

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14 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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Chapter 31

Genetic Engineering and
Biotechnology

Principles Underlying Genetic
Engineering


Biotechnology
: The use of living organisms to
carry out defined chemical processes for
industrial or commercial application. The
modern def. includes
in

vitro

genetic techniques;
manipulation of DNA.


Genetic Engineering
: Techniques including the
isolation, manipulation, and sequencing of DNA,
as well as control of DNA expression, resulting
in genetically modified organisms.

Principles Underlying Genetic
Engineering (cont.)


Premise of genetic engineering is based
on molecular cloning: a DNA fragment
from any organism can be put into and
theoretically made to function in any other
organism.


Developments Essential for the
Development of Genetic Engineering


1.
DNA chemistry



2. DNA enzymology


3. DNA replication


4. Plasmids and conjugation


5. Temperate bacteriophage


6. Transformation


7. RNA chemistry and enzymology


8. Reverse transcription


9. Reglation


10. Translation


11. Protein chemistry


12. Protein excretion and posttranslational mod.


13. The genetic code

Hosts for Cloning Vectors


What are the ideal characteristics of a host that
you’d use for obtaining large amounts of cloned
DNA? What are some actual examples of this
type of host?


What are the disadvantages of these hosts?


What are the advantages of using euk. cells as
hosts?


What is transfection of mammalian cells? Why
don’t we call it transformation?


What are 3 common ways of transfected euk.
cells?

Detecting Clones


Possibilities: Make gene libraries from total
genomic DNA or clone a DNA fragment made by
PCR.


2 situations: 1. Gene of interest is expressed:
detect by complementation, selection, or
antibody


how is this done and what are the
limitations? 2. Gene of interest is not
expressed and you must look for the DNA itself:
nucleic acid probes.

Specialized Vectors


Shuttle vector: cloning vector that can stably
replicate in 2 diff. organisms.


Expression vector: can be used not only to clone
the desired gene, but also contains the necessary
regulatory sequences so that expression of the
gene can be subjected to experimental
manipulation.


Promoters are important in sufficient transcription in
expression vectors. What are ex. of promoters that
have been used in
E. coli
?


Expression vectors must also contain proper
ribosome
-
binding sites (Shine
-
Dalgarno sequence in
prok.).


Specialized Vectors (cont.)


Euk. vectors: Yeasts containing plasmids,
primate DNA virus SV40, mammalian vectors
utilizing adenovirus and vaccinia virus,
integrating vectors: developed so that a cloned
gene can be stably maintained and expressed in
an organism or tissue (low copy #).
Retroviruses can also be used to introduce
genes into mammalian cells since these viruses
integrate into the host chromosome.


Reporter genes: are incorporated into vectors
because they encode proteins that are simple to
detect. What are some ex.?

Expression of Mammalian

Genes in Bacteria (the Problem of Introns)


Use mRNA instead of DNA since mRNA has introns
already removed. The mRNA is used to make
cDNA (complementary DNA copy) using what
enzyme? Can take advantage of poly
-
A tails on euk.
mRNA to purify mRNA (use poly
-
T column).


Can also use RT
-
PCR to synthesize large amounts
of cDNA without having to clone it.


Use reverse translation (protein


mRNA


cDNA)


can make a probe for the actual gene or make a
synthetic gene. Can make modified genes that
have posttranslational processing built in.

Production of Mammalian
Products & Vaccines by GMOs


Microbially produced Human Insulin hormone is
identical in all respects to insulin purified from
human pancreas.


Recombinant vaccines: suspensions of killed or
modified pathogenic microorganisms or specific
fractions isolated from the microorganisms that
cause immunity when injected into an animal
(subunit vaccine), ex. Hepatitis B, measles,
rabies.

Genetic Engineering in

Plant Agriculture


Plant cloning vectors, ex. Ti plasmid from
a gram neg. plant pathogen,
Agrobacterium

tumefaciens
.


Genetic improvement of plants: herbicide,
insect, and microbial disease resistance;
improved product quality.


Other uses: ex. edible vaccines.

Genetic Engineering in Animal
and Human Genetics


Transgenic animals are used to improve
livestock, for research, and to produce
pharmaceuticals, etc.


DNA Fingerprinting: used to ID individuals.


Sequencing of the Human Genome: primary
goal = to understand human genetic diseases.


Gene Therapy: Nonfunctional or dysfunctional
gene is augmented or replaced by a functional
gene, including using viruses as vectors.