Animal transgenesis

Question 1

What is transgenesis?

Answer 1

Cross species transfer of genetic material

Question 2

What makes an animal transgenic? (2)

Answer 2

A genetically modified animal containing a gene from another species. It can be gene addition/deletion or gene modification.

Question 3

What is the point in gene knockout?

Answer 3

To determine the function of specific genes

Question 4

What are the objectives of genetic modification in animal transgenesis?

Answer 4

To produce a stable and heritable change in the genetic makeup of an animal which doesn’t occur under standard conditions. Add/remove genes. Place genes at specifc loci (knock-in). Can also alter the gene expression levels.

Question 5

What are three reasons for modifying animals?

Answer 5

1. Gene function / model disease
2. Modify production traits
3. Organs for xenotransplantation

Question 6

How can disease resistance be a result of modifying animals?

Answer 6

Removal of receptors which make the animal susceptible to disease

Question 7

What is an example of neutraceuticals being produced via modifying animals?

Answer 7

Modification of fatty acids present in meat

Question 8

What are the four methods to produce transgenic animals?

Answer 8

1. Pronuclear injection
2. Embryonic Stem Cells
3. Somatic Cell Nuclear Transfer
4. RNA guided engineered nucleases

Question 9

What is pronuclear injection?

Answer 9

The injection of linearised DNA (gene of interest) (several hundred copies) into either one or both of the pronuclei of the fertilised zygote

Question 10

Why is the DNA linearised and why so many copies?

Answer 10

Multiple copies at random insertion sites, enhancing chances of transgene insertion. Linearised is easier to integrate than circularised, as well as having multiple ends making integration into the genome easier.

Question 11

Which type of cell and why does the transgene need to be inserted into?

Answer 11

Germ cells because these give rise to the founding embryo

Question 12

What two important outcomes come from screening many animals for the transgene?

Answer 12

Determines which animal(s) have the transgene present and in WHICH tissue

Question 13

Following injection, embryos are returned to the surrogate recipient. Mice = 10-20% transgenic offspring, farm animals is 1-10%

Question 14

What are four limitations of microinjection technology?

Answer 14

1. Most animals are not transgenic
2. Variation in transgene expression from founder to founder
3. Mosaicism
4. Flock/herd development is slow

Question 15

What is meant by mosaicism?

Answer 15

Segregation of DNA into different cells, with different expression levels, locations. Hard to determine the impact of the transgene due to complex phenotypes.

Question 16

What are three applications for recombination in mouse ES Cells?

Answer 16

1. Mouse models of human diseases
2. Gene function studies
3. Immunology

Question 17

For gene targeting in mouse ES Cells, what are the first three steps?

Answer 17

1. Design a targeting vector with an antibiotic resistance gene with flanking regions homologous to the gene of interest.
2. ES Cells are isolated from mice embryos/blastocysts and the vector is introduced via electroporation.
3. The wild-type gene already present is replaced by the modified gene (in vector) via homologous recombination.

Question 18

Steps 4-6

Answer 18

4. Antibiotic selection is applied to the ES Cells - only the cells which undergone homologous recombination will survive.
5. Screen clones which were successful through PCR.
6. Inject ES Cells into blastocysts, then implant into surrogate mouse.

Question 19

What type of mouse is produced and why?

Answer 19

Chimeric mouse because it has modified and unmodified cells

Question 20

Step 7

Answer 20

Breed chimeric mice with normal mice to produce offspring with the modified gene.

Question 21

Step 8

Answer 21

Breed the mice continuously until homozygosity is achieved (two copies of the modified gene).

Question 22

What is Somatic Cell Nuclear Transfer (SCNT)?

Answer 22

The nucleus of a somatic cell is transferred into an enucleated oocyte. The resulting cell is stimulated to divide and develop into an organism genetically identical to the donor of the somatic cell.

Question 23

What are four benefits of SCNT?

Answer 23

1. Genetic modification
2. Cloning from adult animals
3. Rapidly produce an unlimited source of donor cells
4. All animals are transgenic

Question 24

100% success in every cell containing the transgene, but 5% are viable to survive. Microinjection have the majority of embryos surviving but few contain the transgene.

Question 25

What are the four main transcription factors responsible for reprogramming of a somatic cell back to a pluripotent state (induced pluripotent stem cell)

Answer 25

IPSC conversion require Oct4, Sox2, Klf4, and c-Myc

Question 26

What are the first two steps in IPSC & SCNT?

Answer 26

1. Adult cells are reprogrammed to a pluripotent state
2. At the same time, an early stage developing embryo is obtained, consisting of only a few cells

Question 27

3 & 4

Answer 27

3. IPSC are introduced into the embryo by injection or incorporation during its culture
4. IPS Cells integrate into the developing tissues and contribute to various tissues and organs

Question 28

What is created now?

Answer 28

Chimeric organism

Question 29

The organism now has cells derived from both IPSC and the host embryo. IPS is not commercial yet. NT has a 5% success rate and microinjection is 10-20% in mice.

Question 30

Why is xenotransplantation using pigs useful?

Answer 30

Similar organ size and it only takes 6 months to produce a pig

Question 31

What are transgenic animals used for? (3)

Answer 31

1. To alter specific traits
2. The production of specific products for pharmalogical applications
3. Used as models for studying diseases and gene function

Question 32

How are transgenic animals used to study diseases?

Answer 32

They can be engineered to carry specific mutations associated with human disease. Study overexpression or knock out of genes. Can test safety and efficacy of therapeutic drugs.