Transgenic animals

Question 1

Define transgenic animal models

Answer 1

Transgenic animal model is an animal that has had a modified foreign piece of DNA inserted into their genome

Question 2

Define transgenic animal

Answer 2

Transgenic animal is any animal that carries a foreign gene (or several foreign genes) deliberately inserted into its genome

= we do this if there is a desirable characteristic

the foreign gene can come from any organism

Question 3

Define transgene

Answer 3

The transgene is the piece of foreign DNA that is inserted into the transgenic animal

Question 4

Why are other sequences of DNA included when the transgene is inserted into the host

Answer 4

In addition to the transgene itself, the DNA usually includes other sequences to enable it

as it enables us to incorporate the transgene into the DNA of the host and to be expressed correctly by the host cells

Question 5

Is it dangerous to insert foreign genes into an animal model

Answer 5

no, because the genetic code is essentially universal.
i.e it is the same in all animals

e.g transgenic sheep and goats express foreign proteins in their milk

Question 6

Why are simple animals used as models

Answer 6

Simple organisms have very simple genomes, they typically do not have backupsystems that humans do, therefore if a gene is taken out we can see its function more clearly

Question 7

Why are mice often used as transgenic animal models

Answer 7

Because they are simple organisms (have fewer genes) therefore the responses can be observed more clearly

and because humans share 99% of their DNA with mice, therefore, we can get more human-like responses from mice

mice are mammals= organ systems similar to humans

mice breed vary easily and quickly for study

Question 8

What does it mean to create a knockout mouse

Answer 8

Creating a knockout mouse

means removing a gene completely from the mouse genome

Question 9

What is the first step in creating a knock-out mouse (infor-wise)

Answer 9

1st step= select novel gene (usually a gene with an unknown function)

2. begin with bioinformatics so that we know more information about our novel gene.

Question 10

Steps of creating a knockout (information-wise_

Answer 10

1. select novel gene
2. use bioinformatics to learn about the novel gene
3. send gene off to be DNA sequenced

Question 11

How do we figure out the type and number of basepairs in our gof?

Answer 11

The sequencing of the novel gene will give us all the basepairs

=the sequencing will tell us
=where the gene is

Question 12

____ tells us Tells us where the exons (coding regions) and introns are in our GOF

Answer 12

sequencing Tells us where the exons (coding regions) and introns are in our GOF

Question 13

Will sequencing indicate if there are splice variants of our gof?

Answer 13

yes,

sequencing will tell us whether there are any splice variants of the gene and where they are located

Question 14

When using DNA sequencing of our GOF, if the novel gene is similar to another gene, what can we confer from this?

Answer 14

If the gene is similar to another gene= can make an educated guess about what its function may be

Question 15

what is the 1st question you need to ask when creating a knockout mouse

Answer 15

1. is this gene essential for survival?

If the gene is essential for survival and we knock it out= the animals will not be born = no use

Question 16

what is the 2nd question you need to ask when creating a knockout mouse

Answer 16

2. How can we remove the gene?

there are 2 ways

1. cut it out completely
   or
2. replace the gene with something else

Question 17

____ are created by deleting a gene or disrupting its function

Answer 17

knock-out mice are created by deleting a gene or disrupting its function by inserting something in its place

Question 18

What is the 1st step in creating a knock-out mouse? (pract)

Answer 18

1. creating a targeting vector

creating a piece of DNA that is targeted towards a specific are of the genome (gof)

Question 19

Mouse genetics =

Answer 19

40 chromosomes

20 diploid pairs

Question 20

how do we knockout the gene in every single body cell?

Answer 20

we remove the gene in the embryo

If we knockout the gene in the embryo it will be knocked-out for the lifetime of that individual

Question 21

Blastocyst has __ layers. what are they

Answer 21

3

1. trophoblast
2. inner cell mass
3. blastocoel

Question 22

define Trophoblast

Answer 22

simple outer layer of cells in the blastocyst

=later forms the placenta

Question 23

define Blastocoel

Answer 23

fluid inside the trophoblast which is in the blastocyst

Question 24

inner cell mass

Answer 24

inner cell mass= embryonic step

cells (pluripotent)

Question 25

give an example of a totipotent cell

Answer 25

fertilised egg

Question 26

__ forms the placenta

Answer 26

trophoblast forms the placenta

Question 27

Define pluripotent

Answer 27

Pluripotent means that the particular cell/cells can become any cell type except for the placenta

Question 28

How do we extract the stem cells?

Answer 28

use a syringe to extract the inner cell mass (stem cells) from the blastocyst.

place stem cells on a petri dish that is lined with media for the stem cells to grow and multiply

Question 29

What must the petri dish have for the cells to grow

Answer 29

petri dish should be lined with media for the stemcells to grow and multiply

Question 30

Once the ESC starts to grow we can ____

Answer 30

Once the ESC (embryonic stem cells) starts to grow we can modify their genome to our advantage

Question 31

We modify our genome using ______

Answer 31

We modify our genome using homologous recombination

Question 32

Describe the process of homologous recombination

Answer 32

Homologous recomination

=there to help us repair our DNA, since DNA breakages occur

2 antiparallel strands of DNA

When a chunk of DNA has been ripped out of the double-strands and are left with untidy ends= end resection occurs, where enzymes work from the 5’ to 3’ direction fixing the

Therfore, the enzys trim off some basepairs of the 5’ ends which results in a 3’ overhang

A near identical sister chromatid and undamaged can be used as a template to repair the DNA

the enzymes come in and cause the 2 undamaged strands to separate so that the broken DNA strands can match up. Therefore, the complimentary basepairs of the undamaged strand binds to the bases of the damaged strand.

This strand invasion bubble will move along and snap back down (to the 5’ end) where to continues again until the DNA is repaired

DNA synthesis, and DNA ligation occurs to fill in any gaps in the strand and to tidyup.

DNA is now repaired

Question 33

DNA is always from _____ direction

Answer 33

DNA is always from 5’ to 3’ direction

Question 34

How do we remove a gene using homologous recombination?

Answer 34

take the sequences that are either side of the gene of interest (double-strand break)

When the gene is extracted, ensure the sequences either side of the gene are also extracted together with the gof

If we are creating a knockout gene, we do not want the sister chromatid making a copy of itself and repairing the extracted gene, therefore, we insert a foreign gene that will be recognised by the cell

Question 35

When are plasmids used

Answer 35

When we are creating a targeting vector we usually use a plasmid

Question 36

Define plasmid

Answer 36

circular piece of DNA usually derived from bacteria

Question 37

Why do we add a reporter gene to the targeting vector

Answer 37

So that when the reporter gene is incorporated into the host genome, the reporter gene will fluoresce => will be able to see if the gene has been knocked out in the original cell

Question 38

What is the next step after the stem cells have grown and multiplied

Answer 38

They are exposed to electroporation

Question 39

Define electroporation and its use

Answer 39

electroporation is an electrical charge that will impact the membrane of cells.

stresses the cells so when the cells are stressed they will open their pores and bring in everything (nutrients) from their environment

Question 40

what happens after the stem cells have grown? (LAQ)

Answer 40

The stem cells are grown on the petri dish and exposed to electroporation.

Add the solution of our vector with the reporter gene thst is surrounded by the 2 sequences on either side of the gof. There is a high chance that the stressed stem cells will take-in the vector.

We can clearly see which cells are successful in taking in the DNA sequences.

The reporter gene will have some sort of tag on it e.g a red tag, so when the cells have taken the sequences they will fluoresce red.

cells with red= successful

Therefore, we can then take out the reporter gene and substitute it with our gof.

therefore, we know which cells have been successful in knocking the gene out of its genome.

Question 41

Homologous recombination is used to insert the gof instead of the reporter gene

Answer 41

Homologous recombination is used to insert the gof instead of the reporter gene

Question 42

What do we do once the knock-out stem cells have grown

Answer 42

select all the stem cells that have knocked-out the gene and grow them on a plate with growth media so they divide.

Question 43

What else do we need for creating a knock-out mouse, if we already have the stemcells with the reporter gene?

Answer 43

We need an empty blastocyst. Host provides the empty blastocyst and we use the embryonic stem cells from another animal.

We inject the knock-ed out stem cells into the empty (inner cell mass) blastocyst. This blastocyst can then be implanted into a pseudo pregnant mouse.

Question 44

When creating knockout mice, what do we need to ensure when selecting the parents, individual, species?

Answer 44

The offspring being produced will have different colours. Therefore, it is important that the knockout stem cells come from 1 colour-organism and the empty blastocyst comes from a diff-coloured organism.

The colour of the offspring will indicate

Question 45

Define chimera

Answer 45

chimera individuals express traits/genes (e.g colour) of both parents/cell-lines

Mix
P1= black
P2=brown
Offspring = mix black and brown

Question 46

What do mice have instead of a uterus?

Answer 46

mice have 2 uterine horns, they don’t have a uterus

Question 47

Briefly describe the whole process of creating a knock-out mous

Answer 47

1. extract stem cells from 1 coloured parent
2. grow the stemcells, expose to electroporation, grow on a media with reporter gene
3. select stem cells that express the reporter gene
4. substitute the reporter gene with gof using homologous recombination
5. grow the knock-out stem cells on growth media
6. use an empty blastocyst from a diff coloured parent
7. inject knock-out stem cells into inner cell mass of host
8. inject the blastocyst into uterus/uterine horns of pesudo pregnant animal
9. observe the offspring (hope it gives us chimera offspring)
10. cross chimera offspring with wildtype = to end

Question 48

What do we expect

Answer 48

We would expect the chimeric x wildtype to produce

heterozygous transgenic mice

1=chr with knockout
1= chr without knockout

Breed heterozygous with heterozygous=

expect

50% heterozygous transgenic
(1chr k.o, 1chr w/k.o)

25% homozygous wildtype (chrx 2 w/k.o)

25%homozygous transgenic (1chr k.o, 1chr k.o)

(1 knockout gene from each parent)

Question 49

Define a homozygous knockout animal

Answer 49

An animal whose parents are heterozygous transgenic with 1 knockout in 1 chromosome each.

The homozygous knockout offspring will contain 1 knockout gene from each parent. 2 knockout genes.

= gene has been completely knocked out

Question 50

Breeding chimeras are AKA

Answer 50

knock-out founders

Question 51

Why is there less success with female chimeric mice being produced?

Answer 51

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