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Coding Terms > TS 1 > Flashcards

TS 1 Flashcards

1
Q

In Canada how is animal use in teaching and research governed?

A

Through the Canadian Council on Animal Care

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2
Q

What is the CCAC?

A

A non-profit organization to enact high standards of animal ethics and care in science throughout Canada

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3
Q

What are institutions funded through tri-council (NSERC, CIHR, SSHRC) required to obtain?

A

A certificate of Good Animal Practice from CCAC

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4
Q

When are animals in research, testing, and teaching acceptable?

A

Only if it promises to contribute to the understanding of fundamental biological principles or to knowledge that can benefit humans/animals

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5
Q

When should animals be used?

A

Only if the researcher’s best efforts to find an alternative have failed

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6
Q

What are researchers with animals required to do?

A

Employ the most human techniques and use the smallest number of animals

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7
Q

What is required for the institution to get certification?

A
  1. Every 3years a panel of scientists, veternarians, and community members from CCAC visit UofC
  2. The panel identifies strengths and weakness and makes suggestions to improve
  3. The UofC must submit a response explaining how they would address the changes from CCAC
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8
Q

Who needs to approve ALL animal care use at the UofC?

A

Animal Care Committee

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9
Q

What does ACC look at?

A
  1. Obsersvational vs. experimental
  2. Wildlife vs. lab animals
  3. Researchers vs. teachings
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10
Q

What does the reporting and training involve?

A
  1. Annual updates detailing the number/type of animals used must be submitted
  2. Animal use protocols must be re-reviewed every 4 years if they are still in-use
  3. All users handling either live animals or tissues at the UofC must complete the IAUTP module
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11
Q

What do the ranking of categories represent?

A

The level of invasiveness

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12
Q

What is level A?

A

Tissues/blood COI such as this class

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13
Q

What is level B?

A

Experiments which cause little or no discomfort to the animals

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14
Q

What is level C?

A

Experiments which cause minor stress or pain for short durations

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15
Q

What is level D?

A

Experiments which cause moderate to severe distress or discomfort such as Dr. Cobb’s lab

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16
Q

What is level E?

A

Procedures which cause severe pain, near, at or above the pain tolerance threshold of unasthetized/conscious animals

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17
Q

What are the 3 R’s?

A

Replace
Reduce
Refine

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18
Q

What is replace?

A

Having alternative to animal use

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19
Q

What is reduce?

A

Using the fewest number of animals

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20
Q

What is refine?

A

Have activities using animals that have been optimized to reduce distress

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21
Q

Why does location matter?

A

The gene’s expression location can tell us the function as well as its relationship with other genes

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22
Q

What is SHH?

A

Is a ligand

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23
Q

What is PTCH1?

A

Is a receptor

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24
Q

What is Bmp7?

A

The gene that is expressed in the webbed hands between the digits and leads to apoptosis

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25
Q

What is the process of SHH binding?

A
  1. SHH is transcribed by a cell containing the gene and is then translated to make the SHH protein
  2. SHH protein (ligand) that binds to the PTCH1 receptor
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26
Q

What is WISH?

A

Whole-mount In Situ Hybridization which involves staining the whole embryos and tells us where in an embryo a given gene is transcribed or expressed also it uses a complementary RNA probe

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27
Q

What is the WISH probe?

A

A complementary RNA probe that will hybridize to the template mRNA sequence in the embryonic cell and it is the complementary dioxigenin (DIG)-labeled RNA probe

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28
Q

How is the complementary dioxigenin (DIG)-labeled RNA probe made?

A

In a test tube by in vitro transcription

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29
Q

What is added to the test tube?

A
  • Template target gene DNA
  • Nucleotides A, G, C and U labeled with DIG label
  • RNA polymerase
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30
Q

What happens where there is a U or a uracil?

A

DIG will bind and will form a large bulky compound

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31
Q

What is the process for the WISH assay?

A
  1. Add DIG-labeled probe to fixed embryos
  2. Corresponding to the gene the probe hybridizes to the complementary mRNA
  3. Detect the probe with the anti-DIG antibody attached conjugated to an enzyme
  4. Enzyme conjugate turns added substrate purple to reveal where and (when) the gene is expressed
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32
Q

What is the anti-DIG antibody?

A

Alkaline phosphatase

33
Q

What is the reporter gene?

A

When this gene is expressed it makes a product that you can detect

34
Q

Where are the places that the reporter gene can be expressed?

A
  1. Along with gene of interest
  2. In place of gene of interest
35
Q

How are reporter genes engineered?

A

Through homologous recombination

36
Q

What is LacZ?

A

A reporter gene that encodes for B-galactosidase

37
Q

What does B-galactosidase do?

A

It carries out a reaction to stain the embryo when the substrate is added

38
Q

What does the location of staining indicate?

A

Where the gene is expressed and its function

39
Q

What happens when a reporter gene is inserted into the coding sequence of a gene?

A

It nullifies it

40
Q

What happens when a reporter gene is inserted in-line with the coding sequence of a gene?

A

If it does not disrupt the reading frame then it is translated with an internal ribosome entry site

41
Q

What happens if it attached to the coding sequence of a gene?

A

It creates a fusion protein

42
Q

What are enhancers?

A

These are known DNA sequences that are cell, tissue, and stage-specific and promote transcription activity by binding transcription factors and each enhancer has different strengths

43
Q

What is a transgenic reporter assay?

A

It uses a reporter gene to show when the enhancer is active

44
Q

What is introduced into an embryo to make a transgenic animal?

A
  1. DNA construct that contains the enhancer sequence
  2. A promoter
  3. A reporter gene
45
Q

How is the enhancer related to the reporter gene?

A

It expresses the reporter gene

46
Q

What does the reporter gene expression correspond to?

A

When and where the enhancer is active
the location of staining => where the enhancer activated LacZ

47
Q

What is a pronuclear injection?

A

This is a method of making a transgenic animal

48
Q

What is the process for pronuclear injections?

A

Use a thin glass pippette to inject the linear DNA into the pronucleus of a fertilized egg

49
Q

What is a pronucleus?

A

This is when the maternal (egg) and paternal (sperm) DNA in the pronuclei are not fused

50
Q

What happens when the linear DNA is randomly incorporated?

A

It can be inserted into an essential gene and kill the embryo or a reporter gene may be activated and lead to abnormal expression somewhere else

51
Q

What happens after the injection?

A

The pronuclei eventually fuse to create a single nucleus for the zygote

52
Q

How many contstructs are incorporated?

A

Multiple copies

53
Q

What is the luciferase assay?

A

This determines if the sequence has enhancer activity or not and quantifies its strength

54
Q

What is a minimal promoter?

A

Allows for the formation of the initiation complex between the coding sequence and the enhancer

55
Q

What is the enhancer of interest added to?

A

Its inserted into a vector that has a minimal promoter and the luciferase reporter gene

56
Q

How is the vector added to cells?

A

The vector with the enhancer of interest is transfected into cells

57
Q

What will the enhancer do?

A

Drive luciferase expression which will be quantified by a luminometer

58
Q

How can we determine the strength of an enhancer and quantify how it changes?

A
  1. Alter the nucleotide sequence of the enhancer and disrupt the transcription factor binding sites
  2. Add or remove transcription factors
59
Q

What is a loss of function mutation?

A

Knockout of a gene

60
Q

What is a gain of function mutation?

A

Overexpression of a gene

61
Q

What is endogneous expression?

A

Where the gene is normally expressed

62
Q

What is ectopic expression?

A

Expression of a gene where it is not normally found

63
Q

What is standard gene knockout?

A

Disrupting the DNA sequence in the gene of all the cells of the organism so it is no longer able to make a protein

64
Q

What causes a standard gene knockout?

A

A DNA construct

65
Q

What is homologous recombination?

A

Where similar DNA sequences are exchanged between the construct and the endogenous gene

66
Q

What is used as a marker in the construct?

A

A gene that encodes for antibiotic resistance

67
Q

What happens after the cells with resistance are selected?

A
  1. The cells are injected in the inner cell mass of the blastocyst
  2. The blastocysts are then implanted into a surrogate and grown
  3. Chimaras are bred
68
Q

What is the difference between a transgenic mouse and knockout mouse?

A

Transgenic:
1. Edits all the cells in the organism
2. Makes a chimara

69
Q

What is conditional gene knockout?

A

If you conduct a standard gene knockout on an essential gene the organism dies

70
Q

What is cre?

A

A viral enzyme that excises genes that are floxed

71
Q

How do you flox a gene?

A

Surround it with LoxP sites on either sides

72
Q

Does floxing a gene disrupt its function?

A

No

73
Q

How is cre expression driven?

A

With cell specific promoter and enhancers

74
Q

What happens wherever the cre is expressed?

A

The floxed genes are removed

75
Q

What is a constituitive promoter?

A

It drives gene expression in all cells constantly

76
Q

How can we use this Rosa26 promoter for a gain of function experiment?

A

Have an extra copy of the gene be expressed

77
Q

What is a stop cassette?

A

Any region of the DNA sequence with labelled with LoxP sites between the promoter and the target gene

78
Q

What does the stop cassette allow?

A

The gene can be conditionally overexpressed because cre will excise just the floxed region

79
Q
A

Coding Terms (17 decks)

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