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Flashcards in Transgenics Deck (42):
1

What is an ES cell?

An embryonic stem cell

2

What are the uses of ES cell chimeras?

Knockouts
Knockins
Genetraps
Conditional gene regulation

3

ES cells are pluripotent, what does this mean?

They contribute to many adult tissues including the germline

4

How can ES cells be transferred back to the embryo?

Microinjection
Aggregation

5

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

6

What are the two ways genes can be targeted in ES cells?

Sequence replacement
Sequence insertion

7

Who derived the first mouse ES cells from a blastocyst and cultured them in vitro in 1981?

Evans and Kaufman
Martin

8

Who cultured the first embryos from and 8 cell blastocyst in 1949?

John Hammond

9

Who defined the exact conditions of in vitro culture of 1 cell blastocyst embryos in 1971?

Whitten and Biggers

10

What are the uses of direct DNA transgenics?

Lineage-specific expression
Reporter transgenes
shRNA
Large fragments of DNA: BAC/PAC (expression of multigene clusters)

11

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

12

Give an example of an application of reporter gene expression in direct DNA transgenics

Expression of lacZ gene in limb buds

13

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

14

What are the three transgenic approaches?

Direct DNA transgenics
Embryonic stem (ES) cell chimaeras
Infection of embryos/ES cells with viruses

15

Outline how direct DNA transgenics is carried out

1. Microinjection of vector into 1dpc mouse oocytes
2. Random integration of DNA into host genome

16

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

17

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

18

What is the infection of embryos/ES cells with viruses used for?

shRNA
Gene expression studies

19

What is talin?

A protein that mediates the attachement of cells to the extracellular matrix and is critical for cell migration

20

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

21

Give an example of an application of gene traps in transgenics

Expression of myosin light chain 2v specifically in the embryonic heart

22

Describe conventional gene deletion

Deleted in all cells
Therefore, if embryonic lethal, cannot derive cell lines or study function in adult tissues

23

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

24

What systems are used in conditional gene deletion?

Recombinase-based systems, e.g. Cre-lox, FRT-FLP

25

What organism is Cre recombinase from?

Bacteriophage P1

26

What does Cre recombinase do?

Catalyses the recombination between tow loxP sequences to form a linear and a circular molecule

27

What is special about loxP sites?

They consist of palindromic repeats flanking a central core

28

Give an example of an application of conditional gene knockout in transgenics

Talin1 conditional gene knockout to cause specific deletion in platelets

29

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

30

What is the most easily-manipulated mammal to use as a model organism?

The mouse

31

What does the use of recombinase systems allow for gene modifications?

For them to be temporally and spatially controlled

32

What does gene targeting undertaken in ES cells allow?

Any endogenous gene to be genetically modified

33

Summarise how transgenics can be made

By direct DNA injection
By use of viruses
By use of ES cells

34

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

35

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

36

Give examples of commonly used inbred strains

C57BL/6
BALB/c
AKR
CBA

37

How many inbred strains have been derived?

~200

38

Around the time that the laboratory mouse originated, who bred and sold mice from her home ~1900?

Abbie Lathrop

39

Who conducted the first breeding experiments for analysis of genetic traits?

Castle
Cuenot
Haldane
Bateson

40

Which species are most laboratory mice established from?

Mus musculus domesticus

41

One of the most important phases in the history of mouse genetics came through the derivation of what?

Inbred strains

42

What engineered nucleases can be used to carry out genetic modifications?

Zinc finger nucleases (ZFNs)
Transcription activator-like effector nucleases (TALEN)
RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)
CRISPR-associated (Cas) nuclease system