CLASS 4.6: 05/01/07 GENETIC ENGINEERING

cattlejoyousBiotechnology

Dec 10, 2012 (4 years and 9 months ago)

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CLASS 4.6: 05/01/07
GENETIC ENGINEERING
A. Recombinant DNA:
1. Cloning
a. Genetic engineering
- Encompasses
i. Manipulations
ii. Introduction
iii. Generation
b. Overview of DNA cloning
- Cloning =
- Two ways that DNA can be cloned (amplified)
i. Place DNA
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ii. Use
- Need
- Must
- Use

a
). DNA

b
). annealing

c
). extension of

- Can use PCR for:
a). Forensics
- ID victim
- place suspects

can use

fingerprints

bodily fluids
b). Basic research
- make
- characterize
- address how
c). Solving historical mysteries
- did
Neandertals
- did Abraham Lincoln
- why
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1918 Spanish Flu Statistics:
-
Infected
-
20-100 million
-
Many victims =
-
Lethal to ~
-
Originated from
- Current bird flu
2. Cloning tools
a. Restriction (
endonuclease
) enzymes
- Identified because
- Bacterial enzymes

recognize


- Work by cutting double-stranded DNA
- Recognition sties
- Often generate
- Molecules cut
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b. Cloning vectors
- Purpose of a vector =
- Works because
- Key considerations
i. Must have
ii. Usually engineered

iii. Usually engineered

- Vectors are limited
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c. Specialized cloning vectors
- Vectors are used
i. Amplify DNA for
ii. Identify sequence with

- Place next to
- Most common reporter
- Isolated from
- Looks green when
- Are several derivatives
iv. Express important proteins
- clone mammalian open reading frame
- can make
- can easily purify
- fewer
immunological
problems
iii. Tag proteins
- can introduce DNA sequence

- must have
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d. Types of recombinant proteins
i. Essential proteins
- examples:
- used to be very expensive
- can be cloned

- must use
- host cells can be
ii. Proteins that can act as drugs
- have normal amount,
- example:
- can be administered
- help fight
iii. Proteins that act as vaccines
- instead of using attenuated or killed pathogens,

iv. Novel proteins
- can block
- act as decoys to
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3. Introducing DNA into cells
- Most eukaryotic cells
- Must use

- Methods of choice
a. Transient
transfections

- Multiple copies
- Cells are induced to take up DNA
- DNA can be expressed,
- Cells should be assayed
- Eventually DNA
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b. Stable
transfections

- Select for events
- Foreign DNA becomes
- Use selectable markers
- Must grow the single cell into a large population =

- Cloned DNA often becomes
- Tandem array becomes
B. Transgenic Organisms:
1. Adding extra copies
- DNA can be
- DNA can be placed
- Multiple copies of a gene
- Usually the endogenous gene
- Expression of the new copies

Expression levels do not

Some genes integrate

Some expressed genes
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- Can make transgenic farm animals
- Can make transgenic crop plants
- Are lots of transgenic plants

Rice that makes

Corn that makes
- Lots of concerns about safety

What will these do

Do no want to place
-
Plants that make their own
pesticide =
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2. Gene targeting and

knockout

mammals
- For research and medical purposes,
a. Gene targeting
- Uses
i. Knock in
- have a mutant
ii. Knock out
- have identified
- want to know
- replace a
wildtype
allele
b. Protocol for gene targeting
i. Modify the gene
- make
- clone it
ii. Introduce the cloned DNA into a specialized cell
type =
iii. Induce a homologous recombination system
iv. Cell will replace
- Extremely rare event (1 x 10
-12
),
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neo
= selectable gene;
c. Embryonic stem cells
- Must make the

- ES cells are
pluropotent
=
- ES Cells are established
- Once it is confirmed that an ES cell has been exactly
targeted
- ES cells that contribute

- Progeny from the animal
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d. Embryonic stems cells and medical research
- Human ES cell lines
- There are approx
- No serious plans
- ES cells have the ability
- Have the potential to cure
i. Type I (juvenile) diabetes =
ii.

Parkinson

s disease =
- Are lots of problems
- NIH-funded labs
C. Molecular Biology of Cancer:
1. Introduction
- Disease in which
- Leads to
- Tumor cells can
- Caused by mutations
- Mutation frequency can be increased
- Not all genes are equal
- Frequency of cancer can
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2. Types of target genes
a.
Proto
-
oncogenes
(c-
oncs
)
- Genes in the cell that
- Often acquire
gain-of-function
mutations

- Mutant form (
oncogene
)
b. Tumor suppressor genes
- Often in genes
- Disease develops when
- Sometimes start with
- Loss of functional allele =
- Examples
i. Genes that control
ii. Genes that
iii. Genes that normally
- when lost,
- BRCA1; BRCA2