Flashcards in Transgenics Deck (42):
What is an ES cell?
An embryonic stem cell
What are the uses of ES cell chimeras?
Conditional gene regulation
ES cells are pluripotent, what does this mean?
They contribute to many adult tissues including the germline
How can ES cells be transferred back to the embryo?
Outline how you would identify ES cells that contain homologous recombination events
1. Generate targetting vector
2. Transfect vector into ES cells
3. Apply selection
4. Pick individual colonies: freeze 80% of colony and prepare DNA from remaining 20%
5. Screen for homologous recombination events by Southern blot analysis or PCR
What are the two ways genes can be targeted in ES cells?
Who derived the first mouse ES cells from a blastocyst and cultured them in vitro in 1981?
Evans and Kaufman
Who cultured the first embryos from and 8 cell blastocyst in 1949?
Who defined the exact conditions of in vitro culture of 1 cell blastocyst embryos in 1971?
Whitten and Biggers
What are the uses of direct DNA transgenics?
Large fragments of DNA: BAC/PAC (expression of multigene clusters)
Give an example of an application of lineage-specific expression in direct DNA transgenics
Melanoma development in mice expressing oncogenic G12D Ras from the tyrosinase promoter
Give an example of an application of reporter gene expression in direct DNA transgenics
Expression of lacZ gene in limb buds
Give an example of an application of shRNA inhibition of gene function in direct DNA transgenics
Inhibition of RAS.GAP generates defects in embryonic and extraembryonic development
Kunath et al., 2003
What are the three transgenic approaches?
Direct DNA transgenics
Embryonic stem (ES) cell chimaeras
Infection of embryos/ES cells with viruses
Outline how direct DNA transgenics is carried out
1. Microinjection of vector into 1dpc mouse oocytes
2. Random integration of DNA into host genome
Outline how ES cell chimaeras are carried out
1. Gene targeting in ES cells
2. Targeted integration of vector into gene locus
3. Microinjection/aggregation of ES cells into 2.5-3.5dpc mouse oocytes
Outline how the infection of embryos/ES cells with viruses is carried out
1. Co-incubation of virus with ES cells or embryos
2. Random integration of virus into host genome
What is the infection of embryos/ES cells with viruses used for?
Gene expression studies
What is talin?
A protein that mediates the attachement of cells to the extracellular matrix and is critical for cell migration
Give an example of an application of gene knockout in transgenics
Talin knockouts die in embryogenesis at E7.5 due to failure of mesodermal cell migration in gastrulation
Give an example of an application of gene traps in transgenics
Expression of myosin light chain 2v specifically in the embryonic heart
Describe conventional gene deletion
Deleted in all cells
Therefore, if embryonic lethal, cannot derive cell lines or study function in adult tissues
Describe conditional gene deletion
Circumvents embryonic lethality
Therefore can delete gene in any tissue and at any time in development or adult
Can derive cell lines for in vitro studies
What systems are used in conditional gene deletion?
Recombinase-based systems, e.g. Cre-lox, FRT-FLP
What organism is Cre recombinase from?
What does Cre recombinase do?
Catalyses the recombination between tow loxP sequences to form a linear and a circular molecule
What is special about loxP sites?
They consist of palindromic repeats flanking a central core
Give an example of an application of conditional gene knockout in transgenics
Talin1 conditional gene knockout to cause specific deletion in platelets
What are the aims of the international knockout mouse/rat consortia?
Knockout every gene in the mouse/rat (~20,000 genes)
Undertake primary phenotype screens
This will allow every gene to be assigned an in vivo function
What is the most easily-manipulated mammal to use as a model organism?
What does the use of recombinase systems allow for gene modifications?
For them to be temporally and spatially controlled
What does gene targeting undertaken in ES cells allow?
Any endogenous gene to be genetically modified
Summarise how transgenics can be made
By direct DNA injection
By use of viruses
By use of ES cells
What is an inbred strain?
One that has been maintained for more than 20 generations of brother-sister matings and is essentially homozygous at all genetic loci, except for mutations arise spontaneously
How would you generate an inbred strain?
1. Outcross to obtain F1
2. F1 x F1 - intercross brother/sister matings
3. F2 x F2 - randomly chisen F2s etc...
4. F19 x F19 - defined as inbred, essentially 98.7% homozygous at all loci
Give examples of commonly used inbred strains
How many inbred strains have been derived?
Around the time that the laboratory mouse originated, who bred and sold mice from her home ~1900?
Who conducted the first breeding experiments for analysis of genetic traits?
Which species are most laboratory mice established from?
Mus musculus domesticus
One of the most important phases in the history of mouse genetics came through the derivation of what?