Genetic Manipulation Technology 1&2 Flashcards
Define pluripotency
The ability of a cell to contribute to any tissue in the body.
Where are pluripotent cells found
Inner cell mass of blastocyst - embryonic stem cells
What is the biggest worry of embryonic stem cells when using them
That they mutate into cancer - forms teratoma in mice if placed under the skin, if placed into mouse uterus - mice formed.
What factor prevents the differentiation of stem cells
Leukemia Inhibitory factor - keep pluripotency
What is Oct4
Gene required for pluripotency -
Transcription factor expressed in inner cell mass cells
What happens if Oct4 is not present
Embryo won’t become blastocyst due to the cells not being pluripotent
What is nanog
Gene required for pluripotency -
What happens if nanog is not present
Loose pluripotency and develop as parietal endoderm
What happens if LIF is removed from ES cells
Embryoid bodies are formed
How genetic manipulation occurs
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
How does desired DNA get into ES cells
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
What is a gene knockout
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
What is random mutagenesis
Point mutations that are caused randomly by a mutagen (e.g. radiation or ENU or EMS) to speed up the process of mutations occuring.
What cells will the mutations occur in when exposed to a mutagen
All cells but the egg/sperm is the most important.
What is a mutagenesis screen
Look at the offspring mice for mutations. Those that are heteroygous for a DOMINANT gene will show a phenotype.
Recessive ones wont
How are recessive mutations screened
Breed the supposed normal mice on for a few generations to see if more mutations occur.
Two benefits of mutagenesis screens
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
Two disadvantages to mutagenesis screens
Uses a large number of animals
Wasteful - only consider ones that are relevant
(Difficult to justify)
What is homologous recombination
Maternal and paternal cross over of chromosones - usually occurs during meiosis
Accidental and quite rare
How is homologous recombination used in knock out genes
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.
Positive negative Selection
Giving cells penicillin resistance and then adding penicillin to kill off the other cells.
What test is done to make sure that the cells have the target vector in them
Southern blot or PCR
3 reasons why some knockouts dont show up straight away in phenotype
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
What are gene knock-ins
Can replace a gene which has been knocked out with a function gene, instead of just a target vector
What is the Rosa26 knock-in
EXTRA READING
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
How well expressed is Rosa26
EXTRA READING
Moderately expressed with its levels varying depending on the age of the mouse.
Also usually presents as single copy expression instead of uncontrollably replicating.
Give some limitations of using mice for gene therapy
EXTRA READING - GRUBB
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
How can stem cells divide independently of leukemia inhibitory factor
EXTRA READING
They can overexpress the Nonog gene through genetic manipulation - also highly present in cancer cells
What did the Luc St-onge et al. 1997 study show regarding Pax6 in mice
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
What did Singh et al 2001 discover regarding targeted deletion of exon 5a in Pax6 of mice
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
What did Ashery-padan et al 2000 discover regarding tissue-specific knockouts of pax6 in the lens
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.
