Transcript P(lacZ,w + )

Training session on
Drosophila mating
schemes
STEP 1: Remind yourself of the key differences
between mitosis and meiosis:
•
crossing-over / interchromosomal recombination during
prophase I (➊)
•
separation of homologous chromosomes during
telophase I (➋)
•
an additional division in meiosis (➌)
Mitosis and meiosis
diploid
generating sister chromatids for each
of the homologous chromosomes
➊
separating
sister
chromatids
➋
➌
haploid
synapsis interchromosomal
recombination
separating
homologous
chromosomes
STEP2: Remind yourself of the basic rules of
Drosophila genetics:
•
law of segregation
•
independent assortment of chromosomes
•
linkage groups and recombination (recombination rule)
•
balancer chromosomes and marker mutations
Law of segregation / linkage groups
Homologous chromosmes are
separated during meiosis
Law of segregation / linkage groups
1
2
1
• each offspring receives
one parental and one
maternal chromosome
• loci on the same
chromsome are passed
on jointly (linkage)
Complication: recombination in females
interchromosomal
recombination takes
place randomly
during oogenesis
Recombination rule:
there is no recombination in males
(nor of the 4th chromosome)
Complication: recombination in females
wildtype
heterozygous
homozygous
mutant
7 instead of 3 different genotypes
Balancers and stock keeping
• lethal mutations are difficult to keep as a stock and will eventually
segregate out
• remedy for work on mice: genotyping of every new generation through PCR
analysis of tail tips - impossible in flies!
Balancers and stock keeping
• lethal mutations are difficult to keep as a stock and will eventually
segregate out
• remedy in Drosophila: balancer chromosomes
Balancers and stock keeping
• balancers carry easily identifiable dominant and recessive markers
Balancers and stock keeping
• balancers carry easily identifiable dominant and recessive markers
• balancers are homozygous lethal or sterile
Balancers and stock keeping
only heterozygous
flies survive and
maintain the stock
• balancers carry easily identifiable dominant and recessive markers
• balancers are homozygous lethal or sterile
• the products of recombination involving balancers are lethal
• During mating schemes, balancers can be used to prevent unwanted
recombination - providing an additional means to the recombination rule.
• You can use balancers and their dominant markers strategically to follow markerless chromosomes through mating schemes.
Rules to be used here:
• 'X' indicates the crossing step; female is shown on the left, male on the right
• sister chromosomes are separated by a horizontal line, different chromosomes are
separated by a semicolon, the 4th chromosome will be neglected
• maternal chromosomes (inherited from mother) are shown above, paternal
chromosomes (blue) below separating line
• the first chromosome represents the sex chromosome, which is either X or Y - females
are X/X, males are X/Y
• generations are indicated as P (parental), F1, 2, 3.. (1st, 2nd, 3rd.. filial generation)
• to keep it simple: dominant markers start with capital, recessive markers with lower
case letters
Now apply your knowledge:
•
follow a step-by-step explanation of a typical crossing
task experienced during routine fly work
•
you will be prompted to make your choices at each step
of the mating scheme; take this opportunity before
forwarding to see a solution
Task: To study the potential effect of a 2nd chromsomal recessive lethal mutation m (stock 1)
on brain development, you want to analyse certain neurons in the brain of m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement insertion P(lacZ,w+) (stock 2). To perform the experiment, you need to recombine
m and P(lacZ,w+) onto the same chromosome. Design a suitable mating scheme.
Tip: w+ on the P-element gives orange eyes.
, Hu
Identify the eye
colours of these flies
Task: To study the potential effect of a 2nd chromsomal recessive lethal mutation m (stock 1)
on brain development, you want to analyse certain neurons in the brain of m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement insertion P(lacZ,w+) (stock 2). To perform the experiment, you need to recombine
m and P(lacZ,w+) onto the same chromosome. Design a suitable mating scheme.
Tip: w+ on the P-element gives orange eyes.
CyO
If
CyO
*
*
Identify all other
markers of these flies
*
TM6b,, Hu
Sb
*
Task: To study the potential effect of a 2nd chromsomal recessive lethal mutation m (stock 1)
on brain development, you want to analyse certain neurons in the brain of m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement insertion P(lacZ,w+) (stock 2). To perform the experiment, you need to recombine
m and P(lacZ,w+) onto the same chromosome. Design a suitable mating scheme.
Tip: w+ on the P-element gives orange eyes.
CyO
If
CyO
Identify the balancer
chromosomes
TM6b,, Hu
Sb
Task: To study the potential effect of a 2nd chromsomal recessive lethal mutation m (stock 1)
on brain development, you want to analyse certain neurons in the brain of m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement insertion P(lacZ,w+) (stock 2). To perform the experiment, you need to recombine
m and P(lacZ,w+) onto the same chromosome. Design a suitable mating scheme.
Tip: w+ on the P-element gives orange eyes.
cross
, Hu
Define the first cross!
Assign ♀ & ♂ to these stocks
Task: Recombine P(lacZ,w+) with lethal mutation m
Selecting F1
stock 2
first?
second?
third?
m
+
;
Y
stock 1
;
Now select
gender and
genotype!
Task: Recombine P(lacZ,w+) with lethal mutation m
Selecting F1
stock 2
first?
second?
third?
m
+
;
Y
stock 1
;
• take females (to allow
for recombination)
• select against curly
wings (to have Pelement & mutation)
Task: Recombine P(lacZ,w+) with lethal mutation m
Designing the F1 cross
+
m
+
haploid
gametes
gonad
Remember: recombination
occurs at random
In the+germline of the
P(lacZ,w
)
selected females,
recombination takes
no recombination
place
*
*
*
*
*
*
m
*
*
P(lacZ,w+)
m
recombination
each layed egg has its individual
recombination history
Challenge: how to select for the
F2 flies carrying correctly
recombined chromosomes?
Task: Recombine P(lacZ,w+) with lethal mutation m
Designing the F1 cross
+
m
+
choose the stock
for the males
Stocks available:
1st step: stabilise recombinant
chromosomes with a balancer
Task: Recombine P(lacZ,w+) with lethal mutation m
F2 selection
m
+
+
first?
F2
second?
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
third?
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
[m]* = potentially
present
+
Sb
not important here;
ignored hereafter
Task: Recombine P(lacZ,w+) with lethal mutation m
Identify the eye colours!
first
F2
second
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
third
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
[m]* = potentially
present
+
Sb
not important here;
ignored hereafter
Task: Recombine P(lacZ,w+) with lethal mutation m
Define your selection
criteria for 2nd and 1st
chromosomes
first
F2
second
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
select for white background, to see orange eyes
third
select
orange eyes,
[m]* for
= potentially
present
for Cy,
against If
+
Sb
not important here;
ignored hereafter
Task: Recombine P(lacZ,w+) with lethal mutation m
Selecting recombinants
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
Challenge: determine whether
recessive "m" is present
Choose female from available stocks
Key strategy: backcross to "m" stock
Task: Recombine P(lacZ,w+) with lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
X
+
m
;
+
CyO
Problem: Each individual represents a unique
recombination event.
Solution: perform many(1) parallel single crosses,
in each using ONE potentially recombinant
male(2) and 3 to 5 females of stock1.
(1) If the chromosomal positions of m and P(lacZ,w+) are
known, the recombination frequency can be calculated;
typically between 20-100 single crosses are required.
(2) Males can mate several females. Even if they die early,
females store enough sperm to lay eggs for a while.
Hence, the likelihood that a single male successfully
establishes a large enough daughter generation is
considerably higher than a single female.
stock 1
Task: Recombine P(lacZ,w+) with lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
F3
All flies Cy?
Canthe
youlethal
spot mutation
the
If yes,
recombinants?
"m"
is present on the
putatively
recombinant
Define your
criteria!
chromosome.
X
+
m
;
+
CyO
stock 1
2nd?
m
P(lacZ,w+),[m]*
m
CyO
CyO
P(lacZ,w+),[m]*
CyO
CyO
To establish the
recombinant stock, you
need to distinguish these
two genotypes, i.e. select
for orange eyes.
Task: Recombine P(lacZ,w+) with lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
X
F3
1st?
m
P(lacZ,w+),[m]*
+
w
+
Y
m
CyO
;
CyO
P(lacZ,w+),[m]*
CyO
CyO
+
m
;
+
CyO
stock 1
Since you have w+
background, this
strategy does not work
To establish the
recombinant stock, you
need to distinguish these
two genotypes, i.e. select
for orange eyes.
Task: Recombine P(lacZ,w+) with lethal mutation m
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
X
Rethink your strategy:
•
given the complexity of genetic crosses, trial and error is
often unavoidable
•
careful planning is pivotal!
Task: Recombine P(lacZ,w+) with lethal mutation m
single males!
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
Choose female from available stocks
aid 1
stock
X
2 possibilities:
• You could use strategy 1, but add a
parallel F1 cross between stock 1
and aid 1 to bring m into w mutant
background (thus preparing it for the
backcross in F2). Try whether it
works for you!
• Here we will use an alternative
strategy, establishing stable stocks
and test for lethality.
to establish stable fly stocks cross
single potentially recombinant
males to the balancer stock
Task: Recombine P(lacZ,w+) with lethal mutation m
establishing stable stocks
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
F3
1st?
2nd?
If
P(lacZ,w+),[m]*
w
w
w
Y
take males
& females
aid 1
stock
X
CyO
P(lacZ,w+),[m]*
;
If
CyO
CyO
CyO
select!
•
•
•
•
for Cy
for orange eyes
against If
against white eyes
Task: Recombine P(lacZ,w+) with lethal mutation m
select stable stocks
F3
w
P(lacZ,w+),[m]*
;
Y
CyO
F4
1st?
X
w
P(lacZ,w+),[m]*
;
Y
CyO
2nd?
All flies Cy?
P(lacZ,w+),[m]*
P(lacZ,w+),[m]*
w
w
w
Y
P(lacZ,w+),[m]*
CyO
;
1
If yes, the marker "m"
is present on the
putatively recombinant
chromosome.
2
CyO
P(lacZ,w+),[m]*
CyO
CyO
1
maintain
as stock
Now continue with independent
crossing tasks