1. Art and Diseño

Mutation verification, allele structure and
the conversion to conditional knockouts
Lydia Teboul
Head of Molecular and Cellular Biology
MLC
Summary
Initial structure of alleles used in IMPC.
Quality control of ES cells and mice: karyotype and allele
structure.
Conversion of KO first into conditional and definitive KO
alleles: tm1a, b, c and d.
Allele structures and phenotypes.
The IMPC allele: KO first
tm1a
loxP
Ex n-1
lacZ
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
The KO first is a genetrap allele with downstream cassettes
flanked by loxPs.
Allele product is a fusion transcript with lacZ allowing for
expression and lineage tracing studies in tissues.
The IMPC allele: KO first
• Promoter driven selection cassette
tm1a
Ex n-1
loxP
lacZ
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
• Promoterless selection cassette
tm1a
Ex n-1
loxP
lacZ
Neo
loxP
Critical Ex (n)
Ex n+1
Either promoter driven and promoterless selection cassettes
have been used to build the collection.
Why are we bothering with QC?
The IMPC ES cell collection was generated in
a high throughput fashion
…therefore materials obtained from repository require
further quality controls of
- karyotype
- integrity of targeted allele
A series of quality control assays are
used to validate ES cells
Karyotyping of ES cells: Switch to ddPCR
JM8 ES cells have been extensively passaged in the process
of generating the mutant collection.
Clones show high frequency of trisomy 1, 8 or 11. Trisomic
clones will not yield germ line transmission.
Trisomy Chr 8
Chromosome spread
ddPCR
Karyotyping of ES cells: Switch to ddPCR
30% of the clones we received in 2014 were trisomic for at
least one of the chromosomes profiled.
Verification of loxP presence
loxP
Ex n-1
lacZ
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
Universal
primers
Gene specific or universal PCR &
sequencing to detect the 3’ LoxP
Gene
specific
Confirm identity of the
targeting construct?
Confirm presence/absence
of downstream LoxP site?
High throughput Southern blotting
n’ kb (lacZ or neo)
m’ kb (neo)
loxP
Ex n-1
lacZ
5’HA
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
3’HA
n kb (lacZ)
m kb (neo or lacZ)
Confirm desired locus
has been targeted?
Confirm structure of
the targeted allele?
Confirm absence of
additional vector
insertions?
QC of alleles in mice: Genotyping by
copy counting
• Full automation of qPCR onto 2
TECAN robots and 384 qPCR
machine.
BP-LOA
NEO
LACZ
CRE
CR LOA
• Copy counting of lacZ/Neo cassette
and WT alleles (loss-of-allele assay).
• Unambiguous allele specific profile:
Any mix-up between stocks or
heterogeneity in line would be
picked up.
Tm1a-het
Tm1b-het
WT
Allele quality: all lines are controlled to
the highest standard
ES cells (2014, ~800 clones)
Mice
Genotyping by allele counting
 Amplification + sequencing target
region
 >99% pass rate for F1 mice
 Southern blotting for imported
stocks
!!! 84% pass rate for imported
lines
Allele conversions:
tm1a
loxP
Ex n-1
lacZ
Flp
loxP
Neo
loxP
Critical Ex (n)
Ex n+1
Cre
tm1b
Ex n-1
tm1c
Ex n-1
Critical Ex (n)
Cre
tm1d
Ex n-1
Ex n+1
Ex n+1
lacZ
Ex n+1
Allele structures and phenotypes
• tm1a and tm1b phenotypes can differ
• Example were tm1a phenotype is less severe than tm1b:
Jmjd5tm (tm1a data from O.Wendling, ICS)
tm1a/tm1a
+/+
tm1b/tm1b
tm1b/+
Leaky gene trap?
-> Rich allele series
Allele structures and phenotypes
• tm1a and tm1b phenotypes can differ
• Example were tm1a phenotype is more severe than tm1b:
Afmidtm
45
40
35
Measure of free fed
glycemia
30
25
20
15
10
5
0
WT
AFMID TM1A
AFMID TM1B
Disturbed spacing or
selection cassette
promoter interferes
with locus function?
Dosage effect?
Summary
tm1a
Ex n-1
lacZ
Neo
Critical Ex (n)
Ex n+1
IMPC alleles are versatile: they can be employed as null or
conditionals.
Genetrap component allows for gene expression and lineage
tracing studies.
The different versions of a targeted locus constitute
potentially rich allelic series.
The large-scale mouse production programme at Harwell
includes a comprehensive quality control of mutated alleles.
We have the capacity of converting KO first into conditional
alleles or definitive KOs on demand.
Thank you for your attention
Molecular Biology Team
Gemma Codner
Adam Caulder
Lauren Chessum
Joffrey Mianne
Jorik Loeffler
Adam Radage
Ted Evans
Colin Beechey
Microinjection Team
Martin Fray
Deb Bogani
Wendy Gardiner
Phenotyping data
James Cleak
Michelle Stewart
Olivia Wendling, ICS, Strasbourg
Questions?