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Transgenic Animals. Flashcards

1
Q

Define CRE-recombinase?

A

A tyrosine recombinase enzyme that is derived from the P1 bacteriophage.

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2
Q

Define floxed DNA sequences?

A

DNA sequences that are between 2 LOX sequences.

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3
Q

Define gene knockout?

A

The removal of harmful or undesired genes.

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4
Q

Define a stem cell?

A

An undifferentiated cell.

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5
Q

Define a target construct?

A

A disrupted version of a gene that will be used in gene knockout.

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6
Q

What is a genetically modified animal?

A

An animal that carries a foreign gene that has been deliberately inserted into its genome.

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7
Q

How is the foreign gene that is inserted into a transgenic animal constructed?

A

By the use of recombinant DNA technology.

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8
Q

Who invented transgenic animals by inserting foreign genes inside the oocytes of Xenopus flies?

A

A scientist called Gurdon.

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9
Q

Who coined the term transgenic animals?

A

Gurdon and Ruddle.

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10
Q

What are some examples of transgenic animals?

A

Transgenic fish such as; Glow-fish.

Transgenic mice; Knockout mice.

Transgenic goats; Silk-milk goats.

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11
Q

What kind of animal is created when 2 different species are bred together?

A

A hybrid.

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12
Q

What is a drawback of the creation of genetic hybrid animals?

A

The hybrids cannot reproduce.

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13
Q

Why can hybrids often not reproduce?

A

As each of the parent species has a different number of chromosomes.

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14
Q

What are some examples of genetic hybrids?

A

The mule. Donkey and horse.

Liger. Male lion and female tiger.

Tigon. Male tiger and a lioness.

Zonkey. A male zebra and a female donkey.

Zedonk. A male donkey and a female zebra.

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15
Q

What 3 categories can the benefits of transgenic animals be placed into?

A

Agriculture.

Industry.

Medicine.

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16
Q

What has the science of creating transgenic animals allowed for?

A

The creation of animals that exhibit specific traits that are beneficial to humans.

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17
Q

How has the creation of transgenic sheep benefited agriculture?

A

They have been engineered to produce larger coats which will provide farmer with more wool.

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18
Q

How has the creation of transgenic livestock benefited agriculture?

A

Transgenic livestock contains more meat on their carcass allowing them to be sold for more money.

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19
Q

Why do transgenic fish often grow larger than wild fish?

A

As they have extra copies of the growth hormone which allows them to grow up to 6 times faster than wild fish..

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20
Q

How does xenotransplantation benefit the field of medicine?

A

It allows for animal organs such as kidneys and hearts to be transplanted into humans.

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21
Q

How can transgenic animals be used to produce pharmecuticals?

A

Pharmaceuticals such as insulin and blood clotting factors that can be obtained from the milk of animals.

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22
Q

How can transgenic animals be used to cure genetic diseases?

A

The normal copy of the gene can be grown in transgenic animals and then inserted into the human genome.

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23
Q

How can transgenic animals be used in genetic research?

A

By providing information into how the genome works by via techniques such as gene knockout or gene insertion.

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24
Q

How can transgenic animals be used in the search for new drugs that target specific genes?

A

These genes of interest are inserted into the animal and this allows researchers to test the drug on the animal.

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25
How can transgenic animals be used in the search for new drugs that target specific diseases?
The genes of the animal can also be altered so that it exhibits a particular disease. This allows us to test the effectiveness of drugs against this disease.
26
How can animals be used in the pharmecutical industry?
They can be used for safety testing and drug screening to ensure that the drugs will work effectively.
27
How can transgenic animals be used to test the safety of certain chemicals?
They can be edited so they have the ability to sniff out substances that may toxic.
28
How can transgenic animals be used as industrial bioreactors?
The genes that code for certain proteins are inserted into the animal. The proteins are harvested from the target cells or from milk.
29
How can transgenic animals be used to produce industrial substances?
Bio steel is produced by transgenic spiders and can be used as a suture material in hospitals or in bullet proof vests.
30
What are 4 major problems with the use of transgenic animals?
The inserted gene may code for more than one product. Many transgenic animals have a low survival rate. Many transgenic animals have breeding problems. Mutagenesis and other functional disorders may arise due to the insertion of these genes.
31
What are 3 major environmental and ecological concerns about transgenic animals
The may outcompete the wild type population. They may alter the natural ecosystem. They may pass their altered genes on to other organisms in the wild.
32
What is the general consensus of releasing transgenic animals into the wild?
That transgenic animals should not be released until the genetic engineering approaches are proven.
33
What are the 3 main methods of producing transgenic animals?
The embryonic stem cell method. The pronucleus method. Retrovirus mediated gene transfer.
34
Wher are the stem cells that are used for the embryonic stem cell method collected from?
The inner cell mass of a blastocyst.
35
What is special about the embryonic stem cells that are used in the embryonic stem cell method?
They are pluripotent which means they can become any cell within the body.
36
What happens in the embryonic stem cell method after the embryonic stem cells have been isolated from the blastocyst?
They are then mixed with DNA that contains the gene of interest. This allows for some of the stem cells to absorb the DNA and become transformed.
37
What happens to the transofrmed cells in the embryonic stem cell method?
They are injected into the inner cell mass of a host blastocyst. This blastocyst is then injected into a foster mother. The mother will give birth to a baby that contains the modified genes.
38
What the pronucleus method based around?
It is based around inserting linear DNA sequences into unfertilised eggs.
39
What is the 1st step of the pronucleus method of creating transgenic animals?
To form the gene of interest that is going to be transferred into the embryo.
40
What is the 2nd step of the pronucleus method of creating transgenic animals, after the gene of interest has been isolated?
The DNA is injected into the male pronucleus which leads to the formation of a diploid zygote.
41
What happens during the pronucleus method of creating transgenic animals, after the diploid zygote has been formed?
Mitosis occurs and the zygote becomes a 2 celled embryo. This embryo is then transferred to a foster mother who will give birth to a transgenic organism.
42
What does retrovirus mediated gene transfer use as cloning vectors?
Retroviruses.
43
What is the first step of retrovirus mediated gene transfer?
The insertion of specific genes into the viral DNA.
44
What happens in retrovirus mediated gene transfer after the genes of interest has been added to the viral DNA?
The virus can insert these genes into any organism that it infects.
45
What are the 2 cell types that are produced in retrovirus mediated gene transfer?
Competent cells that have taken up the gene of interest. Chimera cells that have taken up other genes from the virus.
46
What is the first step in the creation of a transgenic fish?
To insert DNA that codes for an extra growth hormone into a bacterial plasmid.
47
What happens during the creation of a transgenic fish once the plasmids have been created?
The plasmids are inserted into a bacteria. The bacteria then replicates multiple times which produces thousands of copies of the gene of interest.
48
What happens during the creation of a transgenic fish once the bacteria has created the copies of the gene of interest?
These copied DNA is removed from the plasmid and inserted into fish eggs.
49
How can we differentiate between the fish eggs that have taken up the gene of interest and those that haven't in the creation of a transgenic fish?
The eggs that took up the DNA will be much larger than those that did not.
50
What are 4 other techniques that can be used to create transgenic fish?
Microinjection. Sperm-mediated gene transfer. Electroporation. Viral-mediated gene transfer.
51
How does the microinjection technique of creating transgenic animals work?
The gene of interest is injected via the use of a fine, glass needle.
52
How does the sperm-mediated gene transfer technique of creating transgenic animals work?
When the sperm is coated with recombinant DNA.
53
How does the electoporation technique of creating transgenic animals work?
When the eggs are subjected to different electric fields.
54
How does the viral-mediated gene transfer technique of creating transgenic animals work?
The insertion of recombinant DNA via viral infection.
55
What are 4 limitations of gene transfer techniques?
Low germ-line transmission. A variable number of genes are inserted into the organism. No universal insertion site. Genes may be over expressed or under expressed. It is not known whether the insertion of foreign genes will affect neighbouring genes.
56
What does gene targeting involve?
Gene knockout and targeted gene replacement.
57
What is gene knockout?
The removal of undesired genes by replacing them with more desirable genes.
58
When is gene knockout often involved?
When learning about the function of a particular gene. To see the different roles of genes in development.
59
What 3 organisms is gene knockout regularly performed in?
Yeast. Mice. Drosophila.
60
What are knockout mice?
Mice that have had a specific gene removed.
61
What studies are knockout mice often involved in?
As models for genetic disorders in humans. As model organisms to study the different developmental patterns.
62
What 2 things are required during the creation of a transgenic mouse?
Embryonic stem cells from a mouse. A disrupted version of the gene of interest.
63
What is the disrupted version of the gene of interest also known as?
The target construct.
64
How much genomic data does the targeting construct contain?
Around 6-10 Kb of genomic data from the target gene.
65
What are the selectable markers used in the creation of knockout mice?
Thymidine kinase. A neomycin resistance gene.
66
What does the neo-r gene do during the creation of knockout mice?
It gives cells resistance to neomycin.
67
Where is the neo-r gene located on the target construct?
Next to the gene of interest.
68
How is the target construct arranged?
The genes of interest and the neo-r gene are located inside 2 flanking regions. The thymidine kinase gene is located outside of the 2 flanking regions.
69
What are the flanking regions that are located on the target construct?
They are homologous to the sequences on each side of the gene that is being knocked out.
70
What do the flanking regions that are located on the target construct do?
They ensure that the gene is correctly added to the mouse genome.
71
What does the thymidine kinase gene do?
It creates the thymidine kinase enzyme which converts a chemical called gancyclovir into a poison.
72
What is the most likely scenario that will occur when creating transgenic mice?
That the target construct will not be taken up at all.
73
What happens during the creation of transgenic mice if the target construct is not taken up?
The embryonic stem cells will be killed by the neomycin as they will not contain the neomycin resistance gene.
74
What is the 2nd most likely scenario that will occur when creating transgenic mice?
That the gene will be taken up via random insertion. This means that overlaps from each side of the falking sequence have been taken up.
75
What happens during the creation of transgenic mice if the target construct is taken up via random insertion?
These cells will survive exposure to neomycin. They are exposed to gancyclovir and produce the TK enzyme which converts gancyclovir to a toxin.
76
What is the least likely scenario that will occur when creating transgenic mice?
That the target construct will perfectly insert itself so that the cells survive exposure to neomycin and gancyclovir.
77
What is conditional gene knockout?
A version of gene knockout that involves the elimination of a target gene from a single organ.
78
What is the most common form of conditional gene knockout?
The CRE-LOX recombination system.
79
What are the 2 components of the CRE-LOX recombination system?
CRE-recombinase. The LOX-P sequence.
80
What does the CRE-recombinase enzyme do?
It recognises a certain sequence in the genome called the LOX-P sequence
81
What happens when the CRE-recombinase enzyme recognises the LOX-P sequence?
It cuts the LOX-P sequence out of the genome.
82
What about the LOX-P sequence will determine how CRE-recombinase works?
The orientation of the LOX-P sequence.
83
How does CRE-recombinase work if the LOX-P sequences are in the same orientation?
CRE will cut the LOX-P sites and remove the sequence in between before joining the ends back together.
84
How does CRE-recombinase work if the LOX-P sequences are in different orientations (facing each other)?
CRE will cut the piece out and flip it before re-joining it back together. This means that the gene is now backwards and won’t work properly.
85
What is the first step of using CRE-LOX to stop a gene being expressed in the liver cells?
We get CRE-recombinase to be expressed in liver cells.
86
How do we get CRE-recombinase to be expressed in liver cells?
We use a cell that is only expressed in liver cells such as albumin. We take the albumin promoter and merge it with the CRE-recombinase gene. This ensures that CRE-recombinase will only be expressed in the liver.
87
What happens during conditional gene knockout after CRE-rembinase has been inserted into the gene of interest?
This gene can be injected into a fertilised egg. This means that CRE will be expressed in the offspring.
88
During conditional gene knockout, what is the offspring from the mouse that contains CRE-recombinase known as?
The trans activator mouse.
89
What happens in conditional gene knockout after the transactivator mouse has been created?
It is bred to a mouse where the gene of interest is surrounded by the LOX-P sites.
90
In conditional gene knockout what is the mouse that contains the LOX-P sites known as?
As the floxed responder.
91
What happens when the trans-activator mouse is bred to the floxed responder mouse?
The CRE-recombinase from the transactivator mouse targets the sequence in between the LOX-P sites. This causes the gene between the LOX sites to not be expressed.

Molecular Biology. (22 decks)

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