Model Organisms And Genetic Experiments In Mice

Question 1

forward genetics?

Answer 1

phenotype driven
-random mutation
-gives phenotype
-gene identification
-can interpret gene function

identifies gene responsible for certain disease
starting point for gene discovery

Question 2

reverse genetics?

Answer 2

gene driven:
-gene identification
-target mutation into that already known gene
-see phenotype
-can interpret gene finction

confirms role of gene in the disease
can see what phenotypes that gene causes

Question 3

candidate region?

Answer 3

can use pedigree and cytogenic analysis to identify candidate genomic region for disease responsibility

then sequence

Question 4

Heidelberg screen?

Answer 4

in drosophila

treat WT drosophila with EMS (strong mutagen)
cross together flies and backcross to obtain flies homozygous for mutation

can isolate genes related to morphology
can glean many fundamental genes and mechanisms of development

identified Hedgehog (Hh) signalling pathway

Question 5

Hh mutation phenotype

Answer 5

drosophila - embryonic lethal
Hh mutation causes failure of forebrain to separate cerebral hemispheres

mouse- decreases Hh - same forebrain defect - some cases result in cyclopia

Question 6

zebrafish advantage for screening?

Answer 6

obtained easaily in large numbers relatively cheap
easily accessiblr for analysis of embryo during development (in egg as opposed to in mother in mammals)
large numbers of zfish can be studied at same time (mouse embryos are problematic to obtain many at once)

Question 7

how to obtain individual homozygous for mutation

Answer 7

introduce mutagen (eg EMS) to one parent
cross with other parent
obtain some offspring heterozygous for mutation
can backcross mutant heterozygous offspring with mutated parent and some of those offspring will be homozygous mutant

Question 8

mouse as model advantages

Answer 8

-mammalian - similar biology to humans
-record keeping of breeding gives different knwon strains suited for different expetiments
-have inbreeding tolerance so can easily create inbred lines
-small, standard diet and environment - so economical to keep
-quick reproduction cycle - 20 day gestation - oestrus cycle only 4 days so can be fertilised evey 4 days - eady to plan experiments due to frequent window

Question 9

mice genetic similarity to humans?:

Answer 9

orthologous genes in mice and humans share 70-90% sequence identity
higher AA identity in proteins (75-90% as mutliple codons per AA)

highly conserved aspects - ribosomal proteins, nuclear regulatory proteins

host defence ligands and receptors most diverged

Question 10

synteny in mice and humans?

Answer 10

synteny between groups of genes that need to stay together for regulatory and interaction reasons

so chunks of synteny throughout genome though chunks are in different places in each

almost complete synteny of x chromosome in both

Question 11

nude mouse properties:

Answer 11

no thymus due to mutation in one gene
so no mature T cells
used in immunological studies

mutation in this one gene also causes deficiencies in multiple organs including the skin (as well as thymus..)

Question 12

ENU - creating new mutants?:

Answer 12

ENU transfers its ethyl group to O and N radicals in DNA
results in mis-pairing
induces single base pair substitutions in spermatogonial stem cells

each F1 offspring from ENU mutagenised male can carry up to 100 gene mutations (heterozygous in F1)

mutated parental male produces mutated gametes throughout life and can sire multiple mutated litters

Question 13

mutation types:

Answer 13

-missense - AA substitution inn product
-nonsense mutation - premature termination of product translation
-hypomorph - mutant allele that retains some gene function - has lost some but is less severe than loss of function
-antimorph - mutant allele antagonises normal WT gene function
-neomorph - mutant allele acquires new function

Question 14

inbred strain properties:

Answer 14

obtained from >20 sequential crosses between brothers and sisters - leading to complete homozygosity

each strain is uniqur - though individuals within strain very similar

fixed genetic background and high probability of homozygosity - every individual identical (except XX XY differences)

limited genetic variation is useful for analysis of effect of genes of interest - not muddied by individual variance of other genes

can also investigate whether finction of specific gene depnds on genetic background

Question 15

outbred strain properties:

Answer 15

heterozygous - differences in individuals even in same litter

they are vigorous so produce lots of offspring and are easy to maintain
though high individual variance means using them is avoided unless necessary

can be useful for approximating human populations (also outbred)
are often good at maintaining mutants on vigorous background so don’t weaken and die

Question 16

genetic linkage

Answer 16

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

Question 17

recombination frequency

Answer 17

No. of recombinant progeny/no. of total progeny

increases with distance between the two genes on the chromosome

Question 18

analysing new phenotypic variant with congenic strain?:

Answer 18

found mouse with new phenotype
analyse with serial backcrossing

cross mutant mouse with inbred tester strain
causes chimeric chromosomes to form via crossover in F1 onward

repeat backcrossing and select for mice with mutant phenotype
this dilutes the mutant mouse’s DNA with that of the WT tester strain while preserving the gene of interest that causes the phenotype

after 10 backcrossings will obtain strain with only a small interval surrounding the mutant allele - differs from the WT by only this narrow genetic interval

this is the CONGENIC strain
difference from WT tester by only this small interval - sometimes just one gene

Question 19

screening dominant ENU mutations

Answer 19

mate mutated male with WT female
only spermatocytes mutated in male so dont see different phenotype

when crossed with WT female dominant mutations will cause difference in the F1 progeny phenotype

only 1 generation needed to screen

Question 20

screening ENU recessive mutations

Answer 20

need animal to be homozygous for mutant allele
mate mutated male with WT female

parent - no phenotype
F1 no phenotype as only possible to be heterozygous for mutant allele at this point

need to produce additional females with the mutation (heterozygous)

mate G1 males with WT females
will produce heterozygous mutant females

can then mate this with P generation father
this cross give 3 genotypes with regard to allele of interest:
homozygous WT
heterozygous
homozygous for mutant
1:2:1 ratio

Question 21

telling different inbred strains apart

Answer 21

Microsatellite markers
-dinucleotide repeats spread throughout genome
-prone to expansion at each location
-causes variation between strains
-use differences in these markers to telll strains apart

can also do this with differences in SNPs

Question 22

identification of gene responsible for phenotype?:

Answer 22

identify interval in congenic strain in which mutant allele is localised

can use microsatellite markers or SNP polymorphisms characteristic to original strain that had the mutation and tell it’s genes apart from rest of tester strain genome

can then PCR the sequences of the genes contained within this interval (if sequences are known)
and then sequence the PCR products and compare to known WT sequence to see differences

this PCR of interval in congenic mouse strategy can be used to see which DNA sequences stay with the trait after multiple backcrossings

Question 23

how to confirm that identified gene is responsible for phenotype?

Answer 23

congenic strain strategy was used ot identify clock gene in circadian ryth,

introduce mutations into the WT gene and observe if you get same mutant phenotype

e.g. here mutant mouse had abnormally lon period of daily activity which became dysregulated
located interval which contained gene responsible

generated overlapping DNA contiges covering while interval
cloned these contigs into BACs
introduced these BACs into mouse genome
found that only a specific BAC could rescue circadian clock funtion
so could precisely identify which gene was responsible for the mutant phenotype

Question 24

methods to generate transgenic animals?:

Answer 24

Pronuclear:
injection of DNA directly into pronucleus of egg

Transfection of Embryonic Stem cells (ES cells):
-transfect ES cells with DNA , select for genetic change and reintroduce cells into embryo

Question 25

pronuclear transgeneis properties?:

Answer 25

-controlled transfer of genetic info into genome
-DNA can be from any source - different species/synthetic
-large phrnotypic change in one step (no chimeras)
-no reliance on selective breeding

-uses microinjection of purified DNA from plasmid, BAC, or YAC vectord
-can be used to express genes or analyse their regulatory elements

Question 26

method for PN transgenesis?:

Answer 26

Egg cell obtained from superovulating female (induced by hormone injections) that has been mated to stud male
male pronucleus off post mating egg is injected

inverted microscope + 2 micromanipulators
1 MM has pipette holding the egg with negative pressure

optical techniques used to localise pronucleus

other MM has DNA injection needle that can inject picolotres of DNA into egg

many eggs don’t take DNA properly and die

injected eggs implanted in pseudopregnant foster female (induced by mating with vasectomised male)

20 days pregnancy - pups born
can test for transgene in pup by taking sample of tail tissue and using PCR with transgene specific primers on the DNA

Question 27

pronuclear injection peculiarities?:

Answer 27

injected DNA integrates randomly into chromosome

site of integration can influence how the transgene is expressed (position effect)

integration can interrupt an endogenous gene (ie can be mutagenic, ~10% of insertions)

injected DNA can recombine to form concatemers before integrating (so transgene insertions are often multicopy arrays)

injected DNA may be partially degraded (end nibbling) before integration: transgene insertions not always fully intact

Question 28

why is each transgenic line unique?

Answer 28

transgene affected by:
integration site
copy number (multicopy array)
integrity of gene

so each line is unique
conclusions need to be based off analysis of progeny of several founders

Question 29

how is mutated ES cell transgenesis possible?

Answer 29

early mammalian embryogenesis is regulative and can adjust to perturbations
e.g. 2 pre implantation embryos can br combined to produce single animal - aggregation chimera

Question 30

blastocyst structure:

Answer 30

3 parts
-Epiblast - gives rise to animals whole body
-trophoblast - gives rise to placenta (tropho=feeding)
-hypoblast - yolk sac

epiblast +hypoblast form the inner cell mass (ICM) of the blastocyst

Question 31

epiblast ES cell properties?:

Answer 31

pluripotent - can differentiate into cells from all 3 germ layers (ecto-, endo-, meso-derm)

can be derived from epiblast and cultured in vitro

ES cells injected back into clastocyst can contribute to all cell types in adult animal

can differentiate in vitro into all cell types

can give rise to tumours - teratocarcinomas if injected in other locations (e.g. under skin)

need to take ES cells from the epiblast before they differentiate into ecto-/endo-/mesoderm
can retain them in state of pluripotency in vitro - and so can introduce targeted mutations into them

Question 32

process of deriving ES cells from mouse?:

Answer 32

derived from epiblast of blastocyst embryos obtained from uterus of female 3 days post-ovulation

ES cells cultured on plates containing non-dividing fibroblasts called Feeder cells, foetal calf serum, and LIF
modern methods use BMP4+LIF in absnce of foetal calf serum and feeders
this retains ES cells in pluripotent state

Question 33

making a chimera

Answer 33

inject mutated ES cells into blastocyst embryo - forms chimeric embryo
implant chimeric embryo into pseudopregnatn female (see earlier)

chimeric pup is confirmed by coat colour (ES cells derived from agouti strain - implanted into albino strain embryo

interested in male chimeras as they have both X and Y chromosomes

some chimeric males will not have any ES cells contributed to their germ cells
crossing with albino female will give an all non-transgenic progeny

some will have ES cells contributing to their germ - so mating with agouti females will give some agouti progeny that will have the transgene incorporated into all their cells
a fully transgenic animal

Question 34

how to do targeted genetic manipulation on an ES cell?:

Answer 34

need:
-targeting vector with:
>homology arms complementary to sequences in locus you’re trying to disrupt
>Useful to use neo-resistance cassette that allows cells to survive in presence of G418 selection marker -can select for cells that have taken up modification

Question 35

process of correct gene targeting in ES cell by targeting vector?:

Answer 35

correctly targets locus through homologous recombination

a negative selection marker HSV-tk cassette has been excluded from the locus upon integration as it is outside of homology arms (HSV-tk confers sensitivity to Ganiclovir)

so can select for cells which havent targeted properly by negative selection of cells containing HSV-tk - confers (vector has integrated by RANDOM INTEGRATION)
and can select with G418 to select against cells where nothing has integrated (dont have resistance to it

correctly targeted cells have G418 resistance and lack ganciclovir sensitivity

Question 36

overall experimental strategy of gene targeting:

Answer 36

transfect ES cells
grow in vitro
pick colonies

if suitablr RE sites exist in the target locus in animal that will receive transgene, and probes for hybridisation exist outside of the homology arms encompassed region

insertion of vector will make these RE sites further apert and give different sized fragments

can also use long range Taq Pol in PCR
one primer complementary to selection marker
other primer complementary to sequence of the target but outwith homology arm encompassed region
-defined predicted length of sequence amplified if integrated properly
-amplification will not happen if randomly integrated

Question 37

analysing gene expression in transgenic cell using reporter constructs:

Answer 37

can knock in a reporter by having the targeting vector incorporate a e.g. LacZ of GFP sequence

expression pattern of reporter will depend on site of integration
reporter is often designed to replace some of coding sequence
so can end up disrupting regulatory constructs if some unknown ones are there

produces Knock in mouse for reporter construct

Question 38

multicistronic constructs:

Answer 38

add IRES inbetween parts of target vector sequence
lecture not clear but i think it makes it express multiple different proteins (hence multicistronic)
however causes downstream cDNA to be expressed at much lower level than first one
not optimal in all cases

2A peptides are self cleaved peptides and allow expression of different cDNAs at equimolar level (IDK what this really impacts lecturer was not clear :):):):):))

Question 39

localising reporters in transgenic embryos:

Answer 39

can add localisation sites to target sequence to affect reporter localisation

-Nuclear localisation signal (NLS) - directs reporter to nucleus
-Myristoylation or faresylation signal sequences - anchor reporter to cell membrane

other sequences exist to label otehr cell structures (golgi, ER…)

Question 40

homozygous gene targeting?

Answer 40

sometimes need to generate ES cells with homozygous mutation
e.g. when disabling both alleles of a gene is necessary

can have targeting vector carry a construct for one selection marker
and then have another vector carry a different selection marker

select cells for both - both need to have integrated - one in each allele

can also use heterozygous mutant ES cell line to make homozygote
create heterozygous whole transgenic organism as seen earlier
then intercross these heterozygotes to create HOMOZYGOUS KO mouse

Question 41

gene targeting for analysis of gene function:

Answer 41

can use mutant ES cell lines to get reporter construct attached to gene without disrupting function

e.g. analysing spleen development
Hox11 gene incorporated with LacZ reporter construct
staining in areas where/when Hox11 is active
staining goes away when spleen is resolved
so Hox11 is important for spleen development

can also create KOs of genes by disrupting allele in ES cell line and observe phenotype:
all Hox10 paralogue KO in mouse
appearance of additional ribs in lumbar region
(some phenotype when only some of 3 paralogues KO’d)
Hox 10 important for lumbar segment identity in A-P patterning

KO can also reveal redundancy
if KO doesn;t give mutant phenotype but gene is known to have finction there
then redundancy in place