Transcript P(lacZ,w + )

Training session on
Drosophila mating
schemes
This document is one part of a Drosophila genetics training package, the
entire strategy of which is described in detail elsewhere (see link).
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
intra-chromosomal
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
•If lethal
mutations
difficult
to keep
as both
a stock
and
eventually
the mutant
alleles are
(blue
and orange)
were
lethal
in will
homozygosis,
which
of these
genotypes
would fail
segregate
out
(i.e. be replaced
bytowtsurvive?”
alleles)
Balancers and stock keeping
• lethal mutations are difficult to keep as a stock and will eventually
segregate out (i.e. be replaced by wt alleles)
• 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
• With balancers lethal mutations can be stably kept as stocks.
• In mating schemes, balancers can be used to prevent unwanted recombination.
• Balancers and their dominant markers can be used 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 chromosomal 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 when in white mutant background (w on 1st).
, Hu
Do not yet start with the cross. You will
first be asked a couple of questions
Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st).
, Hu
Identify the eye
colours of these flies
Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st).
CyO
If
CyO
*
*
Identify all other
markers of these flies
*
TM6b,, Hu
Sb
*
Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st).
CyO
If
CyO
Identify the balancer
chromosomes
TM6b,, Hu
Sb
Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st).
cross
, Hu
Define the first cross!
Does it matter which stocks
you choose ♀♀ & ♂♂ from?
Task: Recombine P(lacZ,w+) with the lethal mutation m
Selecting F1
stock 2
stock 1
Since you will select females in F1, it
does not matter whether you choose
females from stock 1 or 2. The outcome it
identical for females. Test it out!
Task: Recombine P(lacZ,w+) with the 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 the 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 the 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 the lethal mutation m
Designing the F1 cross
+
m
+
Males from which
stock below?
Stocks available:
1st step: stabilise recombinant
chromosomes with a balancer
Task: Recombine P(lacZ,w+) with the 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 the 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 the lethal mutation m
Define your selection
criteria for 2nd and 1st
chromosomes
choosing males is
preferable for reasons
explained later
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 the lethal mutation m
Selecting recombinants
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
Next task: determine whether the recessive
mutation m has indeed recombined with
P(lacZ,w+) in the chosen males
Choose female from available stocks
Key strategy: backcross to "m" stock
Task: Recombine P(lacZ,w+) with the lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
X
+
m
;
+
CyO
Problem: Each male individual represents a
potentially 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 the lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
F3
X
2nd?
m
P(lacZ,w+),[m]*
How can you identify
the recombinants?
Define your criteria!
m
CyO
CyO
P(lacZ,w+),[m]*
CyO
CyO
+
m
;
+
CyO
stock 1
Task: Recombine P(lacZ,w+) with the lethal mutation m
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
F3
If the lethal mutation "m"
was recombined onto the
P(lacZ,w+) chromosome,
all surviving animals carry
the CyO balancer.
X
+
m
;
+
CyO
stock 1
2nd?
m
P(lacZ,w+),[m]*
m
CyO
CyO
P(lacZ,w+),[m]*
CyO
CyO
If so, establish the
recombinant stock by
selecting for P(lacZ,w+),
i.e. for orange eyes.
Task: Recombine P(lacZ,w+) with the 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
If so, establish the
recombinant stock by
selecting for P(lacZ,w+),
i.e. for orange eyes.
Task: Recombine P(lacZ,w+) with the 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 the 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, first establishing stable
stocks then testing for lethality.
to establish stable fly stocks cross
single potentially recombinant
males to the balancer stock
Task: Recombine P(lacZ,w+) with the 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 the 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 (Suppl. Mat. 4
under this link)