Lecture 9: Transgenic technology

Question 1

What are the broad applications of transgenic technology?

Answer 1

1. Biological and medical research
2. Production of pharmaceutical drugs (protein)
3. Experimental medicine
4. Agriculture

Question 2

What is the purpose of GMO microbes?

Answer 2

E-coli, yeast
To produce recombinant proteins
Research to study cell machinery

Question 3

What is the purpose of GMO fruit flies?

Answer 3

They are widely used in biological research as model organism for studying functions of specific genes because they have a relatively simple genome

Question 4

What is the purpose of GMO fish?

Answer 4

Genetically modify them to produce more growth hormone so that they grow bigger –> consumption

Question 5

What is the purpose of GMO plants?

Answer 5

Transgenic plants have been engineered to possess desirable traits, including resistance to pests or harsh environmental conditions, to improve product shelf-life, and increased nutritional value etc.

Question 6

What are the ways in which crops are genetically modified?

Answer 6

1. Traditional breeding: crossing plants and selecting offspring
2. Mutagenesis: exposing seeds to chemicals or radiation
3. RNA interference: switching off selected genes with RNA
4. Transgenics: Inserting selected genes using recombinant DNA methods.

Question 7

What is the purpose of GMO mammals?

Answer 7

1. Used as experimental models: manipulate the genes to study gene function
2. Create disease models for research and drug testing

Question 8

Why is it important to use transgenic animal models in research, as opposed to cell cultures?

Answer 8

1. Differences in growth characteristics in vivo and in vitro
   - Eg: cells are usually stuck to each other in a 3D fashion and in vitro they are stuck together in 2D, so the cells lose a lot of tissue specific activity because there is communication in vivo which isn’t
   possible in 2D.
2. Genes behave differently when transfect into cells as expression plasmid, because the non coding genes and the proteins still affect the activity of the cell
3. Studying intricate processes and interactions between cells and tissues, influenced by specific genes (proteins), can only be done effectively in whole animals.
4. Higher complex biological function can only be studied and understood in intact animals (e.g. cognitive and behaviour functions

Question 9

What are the limitations in using transgenic animals in research?

Answer 9

1. They are costly and require significant time for development.
2. Certain inquiries, such as understanding the precise molecular mechanisms of drug action, might be better investigated using simpler systems like human cell lines.
3. Some genes play essential roles in embryonic development, making it impossible to create gene knockout animals (resulting in embryonic lethality). However, using conditional knockout (cko) mice can help overcome this issue.
4. The complexity of gene function poses a considerable risk to research projects. Detecting subtle phenotypes could be challenging and may take an extensive amount of time, potentially spanning years or even decades.

Question 10

How do we use animal models in pre clinical drug research?

Answer 10

1. Treat mice with increasing doses until they die then you know the lethal dosage then extrapolate based on weight
2. Study the on and off target effects of the drug and determine which toxic responses may occur

Question 11

What are the approaches for generating transgenic animals ?

Answer 11

1. Pro nuclear injection
2. Embryonic stem cells
3. Nuclear transfer

Question 12

How does pro nuclear injection work to produce a transgenic organism?

Answer 12

1. Inject the DNA into the male pro nuclei of the fertilized egg
2. Transfer the fertilized egg to a recipient female (the female will have to be mated with a vasectomized male so her body will respond as through she actually got pregnant that way)
3. Only a small proportion of the injected eggs will yield a transgenic founder animal, which is usually identified by Southern blotting after birth

Question 13

What are male pro nuclei, and why is the injection of the DNA preferred there?

Answer 13

The sperm enters the egg and at that point, before they fuse, there are two nuclei inside,
and the one from the sperm is the male pro nucleus.
They prefer the male pro nuclei
because it’s larger and its position, which makes it easier to inject inside.

Question 14

In pro nuclear injection, do they just implant one oocyte into the mother or multiple?

Answer 14

Multiple, and 20% of them will survive to term and a fraction of those will be a founder
The success rate is 1.6%

Question 15

In pro nuclear injection, what happens to the foreign DNA once you insert it?

Answer 15

Some injected DNA molecules will be randomly integrated into any one of the chromosomes in about 5-25% of injected embryos

Question 16

In pro nuclear injection, are the founders homozygous for the foreign gene or heterozygous?

Answer 16

Heterozygous because the DNA gets inserted into one of the chromosomes

Question 17

What are founders?

Answer 17

The offspring that carry the desired gene in their germline cells, which means those genes can be inherited by their offspring
(Non founders with the gene have it only in their somatic cells)

Question 18

In pro nuclear injection, how many copies of the transgene get integrated at each site of the DNA in transgenic mice

Answer 18

Usually, multiple copies of transgene becomes integrated at each site –> conchatamer

Question 19

In pro nuclear injection, how many sites does the DNA get integrated into in transgenic mice

Answer 19

In most cases, integration occurs only at a single site on one chromosome in a given embryo.

Question 20

Can we cross two founders to give offspring?

Answer 20

Each founder is a unique species and should not be inter-crossed.

Question 21

Who is the transgenic founder crossed with to produce the animals to study?

Answer 21

1. The transgenic founder is crossed with the wild type mouse
2. The heterozygous offspring produced are intercrossed and the homozygous offspring get chosen

Question 22

What are the applications of transgenic mice?

Answer 22

1. Disease models and drug toxicity testing –> may manipulate the mice to produce specific receptors on the cells to allow for infection
2. Transgenic animals for pharming (e.g. transgenic goats, sheep, cows to produce recombinant proteins in milk).
3. Research (studying gene function by gain-of-function approach

Question 23

How do embryonic stem cells work to produce a transgenic organism?

Answer 23

1. Gene manipulation of an embryonic stem cell via gene addition or gene targetting
2. Injection of the ES cell into a a host blastocyst that will develop to form a chimaera that consists of both host and ES cells.
3. Only chimeric mice in which the germline has arisen from the modified ES cells can become the founder of a transgenic line

Question 24

Which animal is used in producing transgenic organisms from embryonic stem cells?

Answer 24

Embryonic stem (ES) cells are only available in mice and, so far, this technology is limited to this species.

Question 25

How does nuclear transfer work to produce a transgenic organism?

Answer 25

1. Genetic modifications via random gene addition or gene targeting are carried out in cultured somatic cells.
2. The nuclei from the modified cells are transferred to an enucleated oocyte by cell
   fusion before their development in recipient animals.
3. Very few successes have been reported using this method because it’s technically demanding, the survival rate of these animals is very low. Eg: dolly the sheep

Question 26

What is gene targetting?

Answer 26

1. Gene targeting is a genetic technique that uses naturally occurring homologous recombination phenomenon to selectively modify an endogenous gene in the genome of an organism (e.g. mouse)
   2.The method can be used to edit genes to result in gene inactivation, mutation in a targeted manner

Question 27

What are the applications of gene targetting?

Answer 27

1. Analyse loss of function or other mutations in rodents
2. To create animal models mimicking specific human diseases or conditions for drug efficacy or toxicity testing
3. By comparing the traits of normal animals (wild type) to those with a specific gene turned off or mutated using gene targeting, we can gain insight into the function of that gene. (However, if multiple genes in a family perform similar functions, knocking out just one might not show any obvious changes.)
4. To better understand the effects of gene mutations found in hereditary diseases, researchers can recreate these mutations in animals using a knock-in approach, helping to unravel their function.

Question 28

What is homologous recombination?

Answer 28

Homologous recombination is a type of genetic recombination phenomenon that occurs naturally in all forms of life, where nucleotide sequences are exchanged between two identical or similar strands of DNA all the time, but at extremely low frequency.

Question 29

What are the endogenous uses of homologous recombination?

Answer 29

1. Widely used by cells to accurately repair harmful dna breaks that occurs on both strands of dna, so the harmful dna is spliced out.
2. Produces new combinations of dna sequences during meiosis, the process by which eukaryotes make gametes like sperm and egg cells in animals —> genetic diversity

Question 30

What is the process of homologous recombination that occurs experimentally?

Answer 30

1. The targeting construct (DNA casette) contains the marker flanked with homologous regions
2. The target chromosome recognises the homologous regions and exchanges its sequence with the marker sequence

Question 31

What is the procedure done for homologous recombination?

Answer 31

1. ES cells are scraped out from a blastocyst and cultured
2. The targeting vector is constructed and inserted into the cell
3. Positive-negative selection to retrieve the cells with the correct gene recombination, which is then propagated.
4. The ES cells are then injected into blastocysts
5. Blastocysts implanted into mouse
6. The chimeric mice born are mated with normal mice, which give birth to targeted mice

Question 32

Is the process of homologous recombination very common in cells?

Answer 32

No its very rare

Question 33

What is the purpose or irradiated feeder cells?

Answer 33

Provides a sticky surface for the ES cells to stick onto. The feeder cells don’t grow and they give out nutrients too.
(These days you can grow it without feeder cells because they also have disadvantages like viral infections etc.)
slide 30

Question 34

What does the DNA cassette contain?

Answer 34

Disrupted gene X flanked by regions of homology on both sides, a gene tkHSV coding for thymidine kinase, and a neomycin resistance gene in between the gene x coding

Question 35

How does the chemical selection process work to select for cells that have undergone homologous recombination?

Answer 35

1. The gene for neomycin resistance should get incorporated while the gene for thymidine kinase should not
2. Thymidine kinase converts ganciclovir into a triphosphate form that inhibits DNA replication, i.e. cytotoxic for recombinant cells carrying the tkHSV gene
3. Cells are first exposed to neomycin, so the cells that have no recombination at all die
4. Cells are then exposed to gancyclovir so the cells that underwent random insertion die
5. Only the cells that underwent homologous recombination will remain

Question 36

After homologous recombination, how are chimeric mice identified?

Answer 36

1. Furs of chimeras containing tissues derived from both transplanted ES cells and host cells are generated.
2. Distinguished by having sizeable patches of hair of two colours, corresponding to cells derived from host embryo and ES cells

Question 37

please read up on the rest of the slides