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Core Lectures > Transgenics > Flashcards

Transgenics Flashcards

1
Q

What is an ES cell?

A

An embryonic stem cell

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2
Q

What are the uses of ES cell chimeras?

A

Knockouts
Knockins
Genetraps
Conditional gene regulation

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3
Q

ES cells are pluripotent, what does this mean?

A

They contribute to many adult tissues including the germline

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4
Q

How can ES cells be transferred back to the embryo?

A

Microinjection

Aggregation

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5
Q

Outline how you would identify ES cells that contain homologous recombination events

A
  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
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6
Q

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

A

Sequence replacement

Sequence insertion

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7
Q

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

A

Evans and Kaufman

Martin

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8
Q

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

A

John Hammond

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9
Q

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

A

Whitten and Biggers

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10
Q

What are the uses of direct DNA transgenics?

A

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

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11
Q

Give an example of an application of lineage-specific expression in direct DNA transgenics

A

Melanoma development in mice expressing oncogenic G12D Ras from the tyrosinase promoter

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12
Q

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

A

Expression of lacZ gene in limb buds

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13
Q

Give an example of an application of shRNA inhibition of gene function in direct DNA transgenics

A

Inhibition of RAS.GAP generates defects in embryonic and extraembryonic development
Kunath et al., 2003

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14
Q

What are the three transgenic approaches?

A

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

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15
Q

Outline how direct DNA transgenics is carried out

A
  1. Microinjection of vector into 1dpc mouse oocytes

2. Random integration of DNA into host genome

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16
Q

Outline how ES cell chimaeras are carried out

A
  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
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17
Q

Outline how the infection of embryos/ES cells with viruses is carried out

A
  1. Co-incubation of virus with ES cells or embryos

2. Random integration of virus into host genome

18
Q

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

A

shRNA

Gene expression studies

19
Q

What is talin?

A

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

20
Q

Give an example of an application of gene knockout in transgenics

A

Talin knockouts die in embryogenesis at E7.5 due to failure of mesodermal cell migration in gastrulation

21
Q

Give an example of an application of gene traps in transgenics

A

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

22
Q

Describe conventional gene deletion

A

Deleted in all cells

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

23
Q

Describe conditional gene deletion

A

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
Q

What systems are used in conditional gene deletion?

A

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

25
Q

What organism is Cre recombinase from?

A

Bacteriophage P1

26
Q

What does Cre recombinase do?

A

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

27
Q

What is special about loxP sites?

A

They consist of palindromic repeats flanking a central core

28
Q

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

A

Talin1 conditional gene knockout to cause specific deletion in platelets

29
Q

What are the aims of the international knockout mouse/rat consortia?

A

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
Q

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

A

The mouse

31
Q

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

A

For them to be temporally and spatially controlled

32
Q

What does gene targeting undertaken in ES cells allow?

A

Any endogenous gene to be genetically modified

33
Q

Summarise how transgenics can be made

A

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

34
Q

What is an inbred strain?

A

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
Q

How would you generate an inbred strain?

A
  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
Q

Give examples of commonly used inbred strains

A

C57BL/6
BALB/c
AKR
CBA

37
Q

How many inbred strains have been derived?

A

~200

38
Q

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

A

Abbie Lathrop

39
Q

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

A

Castle
Cuenot
Haldane
Bateson

40
Q

Which species are most laboratory mice established from?

A

Mus musculus domesticus

41
Q

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

A

Inbred strains

42
Q

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

A

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

Core Lectures (13 decks)

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