Lecture 13 - Genetic Manipulation of Animals

Question 1

Transgenic plants or Animals?

Answer 1

• Animals become restricted in their potency during development
• plant cells are totipotent
• in most animals the somatic cells and germ cells separate at early developmental stage
• differentiated animal cells normally do not dedifferentiate and recapitulate a full developmental programmme

Question 2

What are 4 methods of creating transgenic animals?

Answer 2

1) Pronuclear injection
- the direct injection of foreign DNA into the pronucleus of a fertilised egg
2) Retroviral vectors
- Retroviruses naturally integrate into the genome of a host (can be used to infect EC or early embryos)
3) Embryonic stem cell manipulation
- The introduction of DNA ino the germline via genetically manipulated embryonic stem cells
4) Nuclear transfer
- introduction of DNA into cultured cells which are then used as a source of donor nuclei for nuclear transfer

Question 3

What is the process of Pronuclear injection?

Answer 3

1) Remove oocyte from oviduct of super ovulated mouse
2) microinject DNA into male pronucleus
3) Transfer to oviduct of psuedopregnant female to implant -> transgenic mouse
4) Mate to wild-type to check for germline transmission (often integrates after 1/2 cell divided-> mosaic mous)
5) Mate hemizygotes to produce homozygotic mice

Question 4

What are transgenic founders?

Answer 4

A transgenic pup produced from microinjection, each founder will give rise to unique transgenic line because

• unique integration site
• unique transgene configuration (e.g. single copy or multiple copies in tandem head to tail arrays)

Question 5

What is the retroviral lifecycle?

Answer 5

1) Entry into cell and loss of envelope
2) Reverse transcriptase makes DNA/RNA and then DNA/DNA double helix
3) Integration of DNA copy into host chromosome
4) intregrated DNA transcribed
5) translation and production of capsid protein, envelope protein, reverse transcriptase, RNA copies
6) Assembly of many new viral particles, each containing reverse transcriptase into protein coats

Question 6

When was the first visable genotypic change in transgenic mice and how was it acheived?

Answer 6

1982 by Palmiter et al
Fertilised egg transformed with recombinant DNa containing
-the structural gene for human growth hormone
-strong mouse gene promoter (metallthionein)

Question 7

What are the features of the retroviral genome?

Answer 7

• LTR on both ends - requireed for integration into the host genome
• packaging sequence
• gag - capsid protein
• pol - reverse transcriptase
• env - glycoproteins in viral envelope

Question 8

What are the advantages and disadvantages of pronuclear injection?

Answer 8

Advantages
-applied to a wide variety of species - mouse, cattle, sheet, fish, poultry
-possible to acheive germ line transmission in up to 40% of microinjected mouse eggs
Disadvantages
-labourious - only one embryo at a time
-in mammals other than mice efficiency = 1%
-no selection

Question 9

What is the process by which retroviral vectors are made?

Answer 9

1a) Make expression vector for viral packaging proteins, gag + pol vector and an env vector
2a) make cell lines expressing packaging proteins
MEANWHILE
1b) remove nonessential functions from the full viral genome, creating a retroviral vector without viral genes
2b) clone gene of interest into vector, creating a third vector containing gene of interest, boardering LTR and packaging sequence
3) Transfect packaging cell line
4) cells express viral proteins as the vector RNA is packaged into infectious viral particles
5) isolate virus particles and infect cells of interest
6) viral RNA converted to dsDNA by reverse transcriptase and integrated into host chromosome
7) viral gene expressed in cells of interest

Question 10

When were the first transgenic mice made and how?

Answer 10

1981

1) Donor female embryo removed, 8 cell embryo infected with pre-implantation embryos with retroviral vector containing the transgene
2) Retroviral integrated into the mouse genome of some cells
3) Reimplanted into surrogate
4) screen mosaic pups for transgene to identify transgenic founder
5) check for germline transmission

Question 11

What are the advantages and disadvantages or retroviral vectors?

Answer 11

Advantages
-takes advantage of natural ability of retroviruses to infect host cells
Disadvantages
-Infects only divding cells
-size limit on insertion 8kb
-retroviral transgenes tend to undergo silencing
-offspring are mosaic and transmission is only possible if integrated into germline cells

Question 12

What are lentiviruses?

Answer 12

class of retroviruses that cause chronic illness in the host e.g. immunodeficiency viruses of cattle, cats and man (HIV)

Question 13

How are lentiviruses unlike retroviruses?

Answer 13

can infect dividing and non-dividing cells

Question 14

What are the adavantages of lentiviruses?

Answer 14

• can infect non dividing cells
• simple to deliver
• high efficiency
• can be injected into the perivitelline space of an oocyte

Question 15

What are some examples of transgenic animals created using lentiviruses?

Answer 15

• transgenic green fluoescent pigs

- ANDi the first transgenic monkey

Question 16

What is the embryonic stem cell method?

Answer 16

the introduction of DNA into germline via genetically manipulated embryonic stem cells

Question 17

How are gene targeted mice generated?

Answer 17

1) DNA introduced by electroporation or chemical treatment in ES mouse blastocyst cells
2) select for integrated cells leading to a pure population of targeted ES cells
3) Targeted ES cells injected into blastocysts
4) Blastocysts implanted into foster mothers
5) Foster mothers give birth to chimeric mice
6) cross chimeric mouse with wt
7) results in fully gene targeted mice and normal mice

Question 18

How is targeted gene disruption acheived at the genetic level?

Answer 18

1) gene targeting vector with selectable marker gene e.g. neomycin resistance gene
2) homologous recombination leads to insertion of gene targeting vector resulting in gene with targeted insertion

Question 19

What are the possible outcomes of targeted gene disruption?

Answer 19

1) gene replacement by homolgous recombination from gene targeting vector to gene leading to targeted insertion
2) random ectopic insertion leading to no replacement

Question 20

How can true selection events be selected for in targeted gene insertion?

Answer 20

use of counter selection

• gene targeting vector contains neomycin resistance gene between LTR and herpes tk gene outside the LTR
• if incorrectly inserted cells are killed by glavciclovir (antiviral medication)

Question 21

What are the advantages and disadvantages of the ES cell method?

Answer 21

Advantages
-works v well with mice, often used for studies of the genetic control of developmental processes and disease using knock out mice
-can maintain ES cell indefinetly in culture
-millions of cells can be transformed and selection can be carried out at the cell culture stage
Disadvantages
-restricted to species in which ES cells that colonise the germ line can be established

Question 22

What is nuclear transfer?

Answer 22

replacement of nucleus of an activated oocyte with the nucleus of a donor cell (somatic cell nuclear transfer SCNT)

Question 23

What is the process of SCNT?

Answer 23

1) cells from animal to be cloned are maintained in the lab so that they do not grow or divide
2) nucleus is removed and fused with an empty egg cell after electric current applied
3) reconstructed embryo grows for 7 days
4) Embryos implanted into surrogate mothers
5) cloned animal born with exact DNA of tissue cell donor

Question 24

When was dolly created and through what procesS?

Answer 24

1996

-nuclear transfer from an adult differentiated mammary cell (mammary epithelial cell)

Question 25

How were Megan and Morag created and when?

Answer 25

1985

-nuclear transfer from cells of early embryos that had been cultured for several months in the lab

Question 26

What was the significance of dolly the sheep?

Answer 26

• possible to generate an individual from a differentiated somatic cell
• this gives rise to offspring that are essentially identical to the founder individual supplied donor nucleus

Question 27

In SCNT, what differences are there between the donor and clone?

Answer 27

• mitochondrial DNA
• increased mutation rate
• shorter telomeres
• epigenetic factors