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The origins of mouse strains and substrains
Agouti (A/A; Tyrp1+/Tryp1+)
Brownish-grey
M. Musculus domesticus
Brown agouti (A/A; Tyrp1b/Tryp1b)
Cinnamon
Substrain Abbreviations
Black non-agouti (a/a; Tyrp1+/Tryp1+)
Black
C57Bl/6
(Tyrc/Tryc)
Albino
Whi
White
BALB/c
Brown non-agouti (a/a; Tyrp1b/Tryp1b)
Chocolate colored
B6D2F1
Dilute brown non-agouti
( / Tyrp1
(a/a;
T 1b/Tryp1
/T 1b; Myo5a
M 5 d/Myo5a
/M 5 d)
DBA/2
Pink-eyed dilution (A/A; Tyrp1+/Tryp1+; Piebald (s/s)
Tyr+/Tyr+; Myo5a+/Myo5a+; p/p)
Irregular white spots
Mutation of Fancy mouse
Mutation of Fancy Mouse (Ednrb)
129
129 strains
AK
AKR strains
B
C57BL
B6
C57BL/6 strains
B10
C57BL/10 strains
BR
C57BR/CD
C
BALB/c strains
C3
C3H strains
CB
CBA
D1
DBA/1 strains
D2
DBA/2 strains
Generating new strains and substrains (i.e. inbred strains)
Most research involving mice entails the use of
inbred strains, not outbred stocks. The advantage
of this is that it enables researchers in different
parts of the world to work to reproduce
experiments carried out in any country.
By definition an inbred strain is generated by
brother sister mating for 20 or more generations
and the foundation stock from for the colony can
be traced back to a single breeding pair.
Once mice have been brother sister mated for 20
generations,
ti
th
then,
on average, 98.6%
98 6% off the
th
genome should be homozygous. Therefore, all
mice within a single inbred strain are both
genetically identical (isogenic) and homozygous
at virtually
y all loci.
If the average mutation rate is taken as 5x 106,
then there is a 90% probability that two substrains
will differ at one or more loci after 16 generations
of separation.
Known mutations on laboratory inbred strains of mice
Embryo implantation in the mouse
Following
g fertilization in
the oviduct, the embryo
undergoes several rounds
of mitotic cell division,
ultimately forming a ball
of cells called a morula.
morula
At the late morula stage,
the embryo enters the
uterine lumen and
transforms into a
blastocyst that contains a
cavity (called blastocoel)
with two distinct cell
populations, the inner cell
mass (ICM) and the
trophectoderm (the
progenitor of trophoblast
cells). Before
implantation, the
blastocyst escapes from
its outer shell (the zona
pellucida) and differentiates to produce additional cell types — the epiblast and the primitive endoderm.
At this stage, the trophectoderm attaches to the uterine lining to initiate the process of implantation. E,
embryonic day.
Isolation of Embryonic Stem (ES) cells
Blastocysts are fertilised,
early developing embryos
that have not yet attached
to the mothers uterus
The blastocyst
y on the left is
c.a. 4 days after
fertilization.
In the blastocyst stage, cells of the inner cell
mass (ICM) are totipotent, i.e. they can
contribute to any embryonic tissue.However,
they have lost the capacity to form extraembryonic membranes.
Blastocysts are isolated from an early mouse
embryo.
Blastocyst
Blastocyst attaches
Bl
h to the
h feeder
f d
layer and the ICM begins to grow
Fibroblast feeder cells
Expansion of the ICM
to give ES cells
Cultured on feeder embryonic fibroblasts.
Different from embryonic stem (ES) cells !!
ES cell lines are derived in vitro to give
pluripotent cells, i.e. not differentiated and
can develop into all tissue types and cells of
an embryo
Each ES cell line must be tested for its
ability to make chimeric mice.
Individual ES cell lines are
tested for their ability to
make chimeric mice
ES transfection/selection, blastocyst injection and germline transmission
ES cell targeting:the process
Homologous recombination refers to the
exchange of DNA fragments between two
DNA molecules at an identical site, which
allows insertion of the transgene to be
targeted to a specific location on the
chromosome.
Undifferentiated embryonic stem (ES) cells
have the potential to differentiate into any
type of cell within the developing organism.
These cells are harvested from a blastocyst
stage embryo and cultured in an in vitro
environment.
Transgene positive cells are identified and
sorted using various selection techniques,
including survival selection (positive-negative
selection) and polymerase chain reaction
(PCR) amplification. The modified ES cells
are then injected directly into a normal
bl t
blastocyst
t embryo.
b
Generation of a congenic line
Cell line
RW4
R1c
J1
D3
AB1
TL1
TC1
W4
C1
E14TG2a
Strain source
129X1/SvJ
129X1/SvJ x
129S4/SvJae
129S2/SvPas
129S7/SvEvBrd-Hprt
129S6/SvEvTac
129S6/SvEvTac
129S6/SvEv
129X1/SvJ
129P2/OlaHsd
PluriStem BALB/C
PluriStem C57BL/6N
PluriStem DBA/2N
Genetic Nomenclature
The gene/locus name serves as a unique identifier as it is
the true unit of inheritance. It’s important because it
identifies the gene as a member of a family (allowing
additional information to be inferred), or not, and can be
used to signify any gene orthologs, for example in human.
A gene symbol must i) be unique, ii) be short (normally 3-5
characters), iii) begin with an uppercase letter (not a
number), followed by all lowercase letters / numbers, iv) be
italicized, v) comprise only Roman letters and Arabic
numbers.
b
By contrast, protein
i symbols
b l use all
ll uppercase
letters and should not be italicized.
The Mouse Genome Database (MGD) serves as a
central repository of gene names and symbols to
avoid use of the same name for different genes or
use of multiple names for the same gene. The Rat
Genome Database serves the same purpose for that
species
species.
A key feature of mouse and rat nomenclature is the
Laboratory Registration Code or Laboratory code,
which is a code of usually three to four letters (first
l
letter
uppercase, followed
f ll
d by
b all
ll lowercase),
l
) that
h
identifies a particular institute, laboratory, or
investigator that produced, and may hold stocks of,
for example, a DNA marker, a mouse or rat strain,
or were the creator of a new mutation.
Examples of Laboratory Codes
J
The Jackson Laboratory
Mit
Massachusetts Institute of Tech
Tech.
Kyo
Kyoto University
Tac
Taconics
Nomenclature for gene targeted loci
Mutations that are the result of g
gene targeting
g
g by
y homologous
g
recombination in ES cells are ggiven the symbol
y
of
the targeted gene, with a superscript consisting of three parts: the symbol tm to denote a targeted mutation, a
serial number from the laboratory of origin and the Laboratory code where the mutation was produced.
Cftrtm1Unc
the first targeted mutation of the cystic fibrosis transmembrane regulator (Cftr)
gene produced at the University of North Carolina
Carolina.
Knock in mutations, in which all or part of the coding region of one gene is replaced by another, should be given
a tm symbol and the particular details of the knock-in associated with the name in publications or databases.
Where there has been a replacement of the complete coding region, the replacing gene symbol can be used
parenthetically as part of the allele symbol of the replaced gene along with a Laboratory code and serial number.
En1tm1(Otx2)Wrst
the coding region of En1 was replaced by the Otx2 gene, originating from the
W. Wurst laboratory.
Gene trap mutations are symbolized in a similar way to targeted mutations. If the trapped gene is known, the
symbol for the trapped allele will be similar to a targeted mutation of the same gene using the format Gt(vector
content)#Labcode for the allele designation.
Gt(bl l Z)15B
Akap12
Ak
12Gt(ble-lacZ)15Brr
a gene trap allele
ll l off the
h Akap12
Ak 12 gene, where
h the
h gene trap vector contains
i a
phleomycin resistance gene (ble) and lacZ, the 15th analyzed in the laboratory
of Jacqueline Barra (Brr).
A transgene
g
is any
y DNA that has been stably
y introduced into the ggermline of mice or rats. Such animals can be
created can be by random insertion into the genome (usually by means of microinjection).
Conditional Gene Targeting: the Bacteriophage P1 System
Conditonal gene targeting. Allows the gene to be
removed at a chosen time or in a chosen tissue by
regulating the expression of Cre-recombinase.
Example A. The first exon of the gene is flanked
by loxP sites, ‘floxed’. If this mouse is crossed
with a transgenic mouse that expresses Cre early
i development
in
d l
t andd in
i every tissue
ti
then
th pups will
ill
be born that have deleted the floxed gene in all
tissues.
Example B. If a liver specific Cre is used, such as
an albumin promoter, then the floxed gene will
only be deleted in liver
liver.
Alternatively, adenovirus that has the Cre cDNA
can be injected into the tail vein of the mouse and
It will localise to the liver where it removes the
floxed allele.
Simplified nomenclature for conditional targeted animals
Tg(Pdx1-Cre)1Agb
a transgenic mouse line (Tg) carrying a Cre-transgene driven by Pdx1 promoter (Pdx1)
This is the mouse line number 1 of this transgenic mouse kind (1) created
by Anne Grapin-Botton (Agb) by insertion.
Cftrtm1Unc
For this example, lets pretend that the first targeted mutation of the cystic fibrosis
transmembrane regulator (Cftr) gene was created by a conditional floxed construct
Cftrtm1Unc :: Tg(Pdx1-Cre)1Agb
This mouse carries both the Cre-transgene
Cre transgene (under the expression of the
Pdx1 promoter) and the conditional allele.
Simplified nomenclature for the laboratory
Cftr+/+
wild-type for both alleles
Cftrflox/+
floxed on one allele and wild-type on the other
Cftrdel/+
gene was deleted on one allele (by Cre; see below) and the other allele is wild-type
Cftrdel/+:: Pdx1-Cre
The Cre-recombinanse will delete the floxed allele in tissues were pdx1 is expressed