Genetic Manipulation Technology 1&2

Question 1

Define pluripotency

Answer 1

The ability of a cell to contribute to any tissue in the body.

Question 2

Where are pluripotent cells found

Answer 2

Inner cell mass of blastocyst - embryonic stem cells

Question 3

What is the biggest worry of embryonic stem cells when using them

Answer 3

That they mutate into cancer - forms teratoma in mice if placed under the skin, if placed into mouse uterus - mice formed.

Question 4

What factor prevents the differentiation of stem cells

Answer 4

Leukemia Inhibitory factor - keep pluripotency

Question 5

What is Oct4

Answer 5

Gene required for pluripotency -
Transcription factor expressed in inner cell mass cells

Question 6

What happens if Oct4 is not present

Answer 6

Embryo won’t become blastocyst due to the cells not being pluripotent

Question 7

What is nanog

Answer 7

Gene required for pluripotency -

Question 8

What happens if nanog is not present

Answer 8

Loose pluripotency and develop as parietal endoderm

Question 9

What happens if LIF is removed from ES cells

Answer 9

Embryoid bodies are formed

Question 10

How genetic manipulation occurs

Answer 10

Take out embronic stem cells and maintain them with LIF in an undifferentiated state –> manipulate them in culture and reintroduce into mice –> let mutated mice breed with wild untouched mice –> produces some natural babies and some wild babies –> get two mutated ones to breed to produce fully genetically modified offspring

Question 11

How does desired DNA get into ES cells

Answer 11

Bathe cells in media containing a solution of DNA –> Pass electric shock through the cells (electroporation) —> this opens holes in the cell membrane to allow the DNA to get in. –> selected cells that have taken up the DNA –> take cells and implant them into mice embryo –> inject embryo in to uterus

DNA cant get initially due to being hydrophilic and cell membrane is hydrophobic

Question 12

What is a gene knockout

Answer 12

To delete genes from animals by homologous recombination in ES cells

Use they ES cells instead of the normal cells and inject into blastocyst of mouse to make knocked out gene mice

Question 13

What is random mutagenesis

Answer 13

Point mutations that are caused randomly by a mutagen (e.g. radiation or ENU or EMS) to speed up the process of mutations occuring.

Question 14

What cells will the mutations occur in when exposed to a mutagen

Answer 14

All cells but the egg/sperm is the most important.

Question 15

What is a mutagenesis screen

Answer 15

Look at the offspring mice for mutations. Those that are heteroygous for a DOMINANT gene will show a phenotype.
Recessive ones wont

Question 16

How are recessive mutations screened

Answer 16

Breed the supposed normal mice on for a few generations to see if more mutations occur.

Question 17

Two benefits of mutagenesis screens

Answer 17

Can generate mutations in tissues without a prior assumption or knowledge about which genes are important
Can generate new alleles that would never have been able to be made deliberately

Question 18

Two disadvantages to mutagenesis screens

Answer 18

Uses a large number of animals
Wasteful - only consider ones that are relevant
(Difficult to justify)

Question 19

What is homologous recombination

Answer 19

Maternal and paternal cross over of chromosones - usually occurs during meiosis
Accidental and quite rare

Question 20

How is homologous recombination used in knock out genes

Answer 20

When the chromosome crosses over - the old gene is replaced with “a target vector” - essentially something that will make it easy to spot when rounding up the cells that have took up the knock out DNA E.g - penicillin resistance.

Question 21

Positive negative Selection

Answer 21

Giving cells penicillin resistance and then adding penicillin to kill off the other cells.

Question 22

What test is done to make sure that the cells have the target vector in them

Answer 22

Southern blot or PCR

Question 23

3 reasons why some knockouts dont show up straight away in phenotype

Answer 23

Redundancy (2 genes doing the same job so the other takes over when one has been knocked out)

Early embryonic lethality e.g. no OCT4 or FGF4

The strain of mouse may affect phenotype

Question 24

What are gene knock-ins

Answer 24

Can replace a gene which has been knocked out with a function gene, instead of just a target vector

Question 25

What is the Rosa26 knock-in

EXTRA READING

Answer 25

A locus where the genetic material inserted (transgene) remained remarkably stable and safe. Rosa26 is found on chromosome 6 in mice and is continually used as a knock-in site

Question 26

How well expressed is Rosa26

EXTRA READING

Answer 26

Moderately expressed with its levels varying depending on the age of the mouse.

Also usually presents as single copy expression instead of uncontrollably replicating.

Question 27

Give some limitations of using mice for gene therapy

EXTRA READING - GRUBB

Answer 27

Genome not identical to humans so dont always get desired results - mice were given deltaF508 mutation which accounts for CFTR gene in CF but mice have no phenotype of pulmonary pathology so it is difficult to use this evidence to understand the deltaF508 mutation

Question 28

How can stem cells divide independently of leukemia inhibitory factor

EXTRA READING

Answer 28

They can overexpress the Nonog gene through genetic manipulation - also highly present in cancer cells

Question 29

What did the Luc St-onge et al. 1997 study show regarding Pax6 in mice

Answer 29

Gene knockout was carried out of Pax6 and the targeting vector LacZ was electroporated in ES cells then injected into chimeric mice and bred with wildtype to produce pax6 heterozygotes.

Showed that -
Homozygotes - no eyes
Heterozygotes - iris hypoplasia, corneal defects, cataract

Question 30

What did Singh et al 2001 discover regarding targeted deletion of exon 5a in Pax6 of mice

Answer 30

Pax6 has an exon called 5a, when this is spliced it forms an isoform of pax6

It was discovered that when removed using cre and loxP –> mice developed iris hypoplasia and retinal defects postnatally, meaning pax6(5a) is NOT required for eye development but must be present to maintain eye tissues postnatally

Question 31

What did Ashery-padan et al 2000 discover regarding tissue-specific knockouts of pax6 in the lens

Answer 31

Used Cre-floxP and found that no lens was formed but retinas were normal patterning therefore - Confirms Pax6 required for lens placode development, but that presence of lens not required to pattern retina.