Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Molecular Genetics > Lecture 19 - Mouse Genetics And Transgenesis > Flashcards

Lecture 19 - Mouse Genetics And Transgenesis Flashcards

1
Q

What is a forward genetic?

A

Is phenotype driven based on a random mutations which alters the phenotype you can then identify the gene and interpret the genes function

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is reverse genetics?

A

This is driven by genes and begins with a cloned gene of interest then you study the role of it by mutating it this is how you interpret gene function

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is gene trapping?

A

Where you randomly insert mutagens which introduce a tag do that the mutated gene can be identified before needing mice.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Is gene function conserved among species?

A

Yes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Pax6 causes what phenotype and is this conserved across species?

A

They create eyes and are conserved

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

You can use forward genetics to do what and reversal genetic to do what?

A

Find out what gene is causing a disease and reversal genetics confirms the role of the gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How would you identify a mutated gene from a disease?

A

Forward genetics (predigree analysis and genetic linkage), reversal genetics (cytogenetic analysis) which would then give you a candidate genomic region. You would then select for specific genes and do a candidate gene mutation analysis in those affected before a gene mutation functional analysis (mutate the normal gene which is cell based before putting it into mice or fish)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Drosophila - how is forward genetics done?

A

X-rays, chemicals or transposing are used to make mutant red flies which is used to try and see what gene causes this

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Drosophila - how is reversed genetics done?

A

Discover gen function through targeted disruption/ inhibition of the gene and then analyse the resulting phenotypes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Why are reverse genetics and forward genetics used?

A

Creation of animal models of disease

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Heidelberg screen - what did they do?

A

Engineered a fly with a balancer chromosome, this contained a marker gene and a lethal recessive allele.

They then put a mutagen (EMS) into wildtype fly to create mutations. They crossed the animals and got many phenotypes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is a balancer chromosomes?

A

Engineered through chromosomes inversions and cannot undergo crossing over, they contain lethal genes and markers

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What gene did the heidelberg screen identify?

A

Hedgehog

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

In the mutated flies what did hedgehog signalling cause?

A

Failure in forebrain defects - separation of cerebral hemispheres

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What happens in mice with loss of hedgehogs and humans with the disease HPE?

A

Failure of forebrain defect - the same as in flies

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is the mutagenic agent used in zebra fish screening?

A

ENU

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Why would you use zebra fish in large scale genetic screens?

A

They have offspring quickly, they are also laid in eggs so easily accessible, there is many offspring and are cheap

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are some mutations identified through zebra fish screening?

A
  • One eyes pinhead mutation leading to defects such as cyclopia
  • Weissherbts mutations leading to hypochromic blood with decreasing blood cell counts.
  • Hagoramo mutations leading to a disrupted stripe pattern of adult fish
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Advantages of mouse model?

A

Mammal
Small
Standard diet and environment
Reproductive efficiency - rapid non seasonal breeding
History of careful breeding and record keeping
Tolerant to inbreeding
Large numbers of mutants - spontaneous and induced
Embryo and sperm cryopreservation
Clones DNA and cDNA resources
Sequence genome
Transgenesis
Embryonic stem cells - directed mutagenesis
Level of public acceptability

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are the key parameters of mouse reproduction?

A

Can be pregnant again immediately after parturition
Breed all year round
Embryos and sperm can be cryopreserved
Oestrus cycle - 4 days meaning they can be fertilised every 4 days

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What are some similarities between humans and mice?

A

Genome size,
92% shared genes in the genome
2 sex chromosomes x and Y
They have similar ribosomal proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

Differences between humans and mice?

A

Humans have more autosomes than mice
Humans have meta centric and sub meta centric chromosomes whilst mice only have telocentric
Humans have more CpG islands than mice

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

Why do large chunks of genes need to be together?

A

To interact and be correctly transcribed

24
Q

In humans and mice what chromosomes nearly has complete synteny?

A

X

25
Q

What happens to mice with no thymus and therefore no t lymphocytes?

A

They’re balds

26
Q

How do you create new mutants with ENU?

A

ENU transfers its ethyl group to oxygen and nitrogen radicals in DNA resulting in mis-pairing. This induces single base pair substitution in spermatogonial stem cells at high efficiency do each offspring from males could have hundreds of mutations.

27
Q

What mutations can ENU cause - what is missense and nonsense?

A

Missense - amino acid substitution
Nonsense - premature termination of protein

28
Q

What mutations can ENU cause - what is hypomorph?

A

Hypomorph - a mutant allele that retains some gene function and is less severe than loss of function.

29
Q

What mutations can ENU cause - what is antimorph?

A

Antimorph - a mutants allele which antagonises the normal gene function

30
Q

What mutations can ENU cause - what is neomorph?

A

A mutant allele that requires a new function

31
Q

What happens to the genes of an out red mouse?

A

May contain different combinations of alleles for every gene

32
Q

What happens to the gene in an inbred mouse?

A

Each animal is identical and homozygous for alleles of each gene

33
Q

Advantages of using outbred mouse strains?

A

Approximates human population
Easy to maintain due to heterozygous vigour
Often maintain mutations on vigorous outbred background so don’t weaken and die

34
Q

Advantages of inbred mouse strains?

A

Each inbred strain unique
Made by brother and sister mating for 20 generations at each locus
Fixed genetic background - every individual genetically identical except for xx or xy

35
Q

Why would you use inbred strains?

A

Limited genetic variation - you know what the genes are
Identification of genes responsible for a certain phenotype
Investigation of the role of genetic background in modifying action of genes of interest

36
Q

Genetic mapping

A

Shows the genetic in meiotic divisions - who will inherit what, what has swapped.

37
Q

What is linkage?

A

When two different alleles are transmitted to offspring more frequently in parental combinations than in non-parental combinations

38
Q

What is recombination frequency?

A

Increases with increasing distance between 2 different alleles on the chromosomes

39
Q

how do you calculate recombination frequency?

A

A/N

A = number of recombinant progeny
N = total number of progeny

40
Q

How do you work out what gene is responsible?

A

Serial back crossing to inbred tester strain - introduces the trait locus into a defined genetic background to create a congenic strain

41
Q

How do you construct a congenic strain?

A

Heterozygosity elimination occurs in regions not linked to the mutant locus. Selection of progeny based on mutant phenotype. You keep selecting for mutants during the backcrossing but as you do this you will get a smaller section of mutations until it is only one gene.

Linkage retains significant lengths of flanking chromosomes from the original mutant strain.

42
Q

Is one generation breeding screen sufficient to see a dominant mutations?

A

Yes

43
Q

What would you need to do to find a recessive mutations?

A

Three generation breeding

44
Q

How do you do a three generation breeding screen?

A

Each G1 offspring represents a gamete-sampled from the mutagenised male

A pedigree is established from individual G1 males by mating to a wildtype female

G2 females are then mated back to G1males to produce G3 offspring

G3 offspring screened for variation

45
Q

How do you detect an accurate position of the mutation?

A

Micro satellite markers

46
Q

What are micro satellites?

A

repetitive repeats throughout the genome. Each location the number of repeats is prone to expansion or reduction, leading to variation between mouse strains. We have mapped these and can bee identified by PCR.

47
Q

In practical terms how do you identify genes responsible for phenotypes?

A

Identify the mutation localisation and clone the mutant mouse with a wildtype. You will then get a region in the offspring with the mutations which can be mapped by micro satellites or SNP’s from original mouse strain.

48
Q

What are the flanking markers which can identify specific regions on a mutant strain?

A

Micro satellites
SNPs

49
Q

How do you identify a candidate region?

A

Genetic mapping and then do a PCR for genes and sequence these products

50
Q

Instructions to detect nucleotide level mutations?

A

1) design primers for PCR
2) Amplify target DNA regions by PCR
3) Determine nucleotide sequence of PCR products
4) compare sequences from control and affected individuals.

51
Q

Real example - how did they locate the cycradian clock gene?

A

Micro satellites localised mutation to non-recombinant interval to allow identification of candidate genes.

Construction of BAC contigs and their sequences - chromosome walking: rescue experiments and identification of mutations. In this case it was a rescue with BAC54 only identified in the clock gene, responsible for mutation which gave circadian phenotype.

With completion of mouse genome sequencing project – known DNA sequence of the candidate interval can be used to check mutations in the mutant animal. However, further analysis would be required to confirm that found mutation is responsible for phenotype – like in this experiment

52
Q

What are DNA contigs?

A

Large overlapping genes

53
Q

High throughput (next generation) sequencing - what increases the speed of identifying a mutant?

A

Complete mouse genome sequencing

54
Q

High throughput sequencing - is exome sequencing cheap and sometimes sufficient?

A

Yes

55
Q

Do complete mouse genome sequencing and exome sequencing need further verification?

A

Yes

Molecular Genetics (20 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions