Chapter 31


14 déc. 2012 (il y a 8 années et 7 mois)

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Chapter 31

Genetic Engineering and

Principles Underlying Genetic

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


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

Developments Essential for the
Development of Genetic Engineering

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

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

What are 3 common ways of transfected euk.

Detecting Clones

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

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

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

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
binding sites (Shine
Dalgarno sequence in

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



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,

Genetic Engineering in

Plant Agriculture

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


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.