Micro 2 Lecture 10 Pt. 2

Front 1

Microbes synthesizing a wide variety of proteins used in medicine, agriculture, bioremediation, industry, etc.

Back 1

Recombinant microbes

Front 2

Plants that synthesize vitamins, medicines or agriculturally important substances

Back 2

Transgenic plants

Front 3

Animals synthesizing medicines or providing models for human disease

Back 3

Transgenic animals

Front 4

How is the use of recombinant technology useful in human disease studies?

Back 4

Since cystic fibrosis is only found in humans, it was impossible to use animal research studies. Now, the gene for cystic fibrosis can be placed into animals, allowing us to do animal studies.

Front 5

Creating transgenic plants depends on a certain soil bacteria from the genus:

Back 5

Agrobacterium

Front 6

What is the name of the plasmid that Agrobacterium has?

Back 6

Ti plasmid

Front 7

What makes Agrobacterium interesting?

Back 7

He normally transforms plant cells; he pushes his plasmid (which contains all the genes for making his favorite food source) into plant cells to get them to produce food for him.

Front 8

Ti stands for?

Back 8

tumor inducing

Front 9

Describe what Agrobacterium does?

Back 9

He comes up to a plant cell and pushes his plasmid (Ti plasmid) into the plant cell. The Ti plasmid encodes all the info for making Agrobacterium's favorite food source. The plant begins to produce the food that he likes to eat. As this process continues, he begins to reproduce through binary fission and cause a tumor on the plant.

Front 10

How is Agrobacterium useful to us then?

Back 10

Since Agrobacterium normally injects his plasmid into plants, all we have to do is insert our gene of interest onto his plasmid and he will get the plant to express the gene for us.

Front 11

Describe the process of using Agrobacterium to get a tomato plant to express the human insulin gene:

Back 11

1) isolate and amplify the human insulin gene using PCR & good primer design.
2) Cut hole in Ti plasmid and insert insulin gene, using ligase to glue them together.
3) Place Ti plasmid and human insulin gene into test tube.
4) Encourage transformation by applying a series of chemical treatments, or electricity.
5) Infect the tomato plant by applying Agrobacterium to the side of the stock of the plant.
6) Agrobacterium will inject the plasmid into the plant.

Front 12

What kinds of plants do Agrobacterium infect?

Back 12

Grass plants: tomato, potato, banana, tobacco.

Front 13

What are two real world examples of transgenic plants created with Agrobacterium?

Back 13

tobacco with herbicide resistance gene inserted with the help of Agrobacterium.

Peas with a gene inserted by Agrobacterium that blocks weevil digestion.

Cholera vaccine in transgenic potato plant inserted using Agrobacterium

Front 14

steps for creating a transgenic animal:

Back 14

1) collect embryos from sacrificed female
2) inject gene into nucleus of the embryo
3) success rates are low; it is easy to insert the gene into the nucleus but getting the
gene to be incorporated is another matter.
4) If embryo successfully takes gene, you place the embryo inside a female.

Front 15

A way to repair or replace a patient's faulty gene.

Back 15

gene therapy

Front 16

Describe the steps used the first time a gene therapy technique was used.
First, what are the agents involved?

Back 16

Genetic donor = Normal human immune gene
Vector = virus
Cloning host = little girl

a) a normal immune gene is isolated and amplified using PCR and good primer design
b) place gene of interest into empty virus capsid
c) take cell samples from patient and infect them with the virus vector
d) grow them for a few generations and then return them to the patient

Front 17

Do plasmids vectors work well for transgenic animal techniques?

Back 17

no, they don't work very well for animal cells so they are rarely used.

Front 18

What works better than plasmids for transgenic animal techniques?

Back 18

virus vectors

Front 19

is gene therapy permanent at this point?

Back 19

no

Front 20

What brought gene therapy to a halt in 2000? What is being done now?

Back 20

the death of 2 men because the virus vectors used weren't completely void of viral DNA. Now, viral vectors are deemed too dangerous and alternative vectors are being sought.

Front 21

What are some ideas for alternative vectors being researched now?

Back 21

-the gene gun (shoots gene pellet inside cell)
-liposome vectors (vesicle with gene inside)

Front 22

techniques for analyzing a patients genome:

Back 22

DNA fingerprinting & microarray analysis

Front 23

What year was the human genome mapped? What are some surprising facts that were learned?

Back 23

2001;
-that we share 80% identical DNA with mice and 60% identical DNA with rice plant;
-that there are large sections of the human chromosome that are non-coding: introns

Front 24

What % of the human genome is made up of introns?

Back 24

as much as 80% of the genome is made up of non-coding DNA: introns

Front 25

differences between human intron DNA sequences:

Back 25

polymorphisms

Front 26

Random mutations occur everywhere in the genome but mutations in ________ are more likely to be successfully passes through generations.

Back 26

INTRONS are more likely to be successfully passes through generations.

Front 27

What % of exon DNA is identical among humans?

Back 27

exons = 99.9%

Front 28

Explain why more mutations would be found in introns than exons in the context of natural selection:

Back 28

mutations in introns are free to be passed from one generation to the next, while those in exons are under selective pressure and less likely to be passed to the next generation.

Front 29

Explain the technique used in DNA fingerprinting in general and for criminal forensics:

Back 29

-use restriction endonucleases to make cuts at a specific sequences known
to be identified with people of certain races or regions of the world and run
on gel electrophoresis
-expose victim's and suspect's DNA to the exact same restriction
endonucleases and run on gel electrophoresis. Compare the fragments
in the lanes on the gel.

Front 30

How accurate can DNA fingerprinting be?

Back 30

1 in 1,000,000, and greater

Front 31

transcription & translation
-which genes are being turned on and which are being turned off

Back 31

expression

Front 32

Define microarray analysis:

Back 32

Microarray analysis: Determining the expression of a gene by looking at the mRNA present in a cell.

Front 33

What does microarray analysis allow you to investigate?

Back 33

-health vs disease aspect of a patient: you could compare a healthy cell to a cancerous one and research which genes are
being express and which ones are not being expressed.

-growth vs differentiation: you could look at the development of growth of a patients cells as they move from one phase of their cycle to another.

Front 34

What is the hope for microarray analysis?

Back 34

to improve the accuracy of diagnosis and treatment.

Front 35

If a kidney cell and an eyeball cell in your body both have the exact same DNA, what makes them different?

Back 35

kidney cells only express kidney proteins.
eyeball cells only express eyeball proteins.

The importance isn't in the DNA, it's what genes are being expressed and which ones aren't being expressed.

Front 36

Describe the steps in microarray analysis:

Back 36

1) Isolate mRNA from a patient's cells and wash it over a disk that has all human genes (31,000) attached to it.
2) The mRNA will bind to its complementary gene on the disk. Wash off the excess and place it into the microarray reader.
3) The microarray reader identifies which genes are being over expressed or under expressed.