Genetics

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Genetics and Breeding

LAT Chapter 4

Chapter 4


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Chapter 4


Genetics


To breed laboratory animals successfully,
basic knowledge of genetics and reproduction
is required.


The breeding system selected must meet the
requirements of the research program for
which the animals are being bred and must
correlate with the behavioral characteristics of
the species.


This chapter focuses on basic genetic
concepts as they relate to breeding colony
management.




Chapter 4


Heredity


Genetics is the science of heredity.


Hereditary characteristics are determined by
units called genes, carried on chromosomes.


Genes are transmitted from one generation to
the next, through asexual reproduction, or by
sexual reproduction.


Genes are found in cell nuclei and are
composed of DNA.


Every characteristic of an organism, from hair
color to heart size, is determined by the genes it
received from its parents.

Chapter 4


Dominant and Recessive Alleles


2 sets of chromosomes, 1 from each parent.


Each gene on 1 chromosome has a partner at
the same locus, on the matching chromosome
of the set.


All genes at the same locus are called alleles.


A dominant allele excludes the expression of a
recessive allele.


A recessive allele express itself when 2
recessive alleles are present.


More than 2 alleles common for the same trait.

Chapter 4


Gene Symbols


To facilitate prediction of what the offspring from
the mating of two animals will look like, letters
are used to represent different genes and their
alleles.


Capital = dominant / lower case = recessive.


Be exact and accurate when recording gene
symbols.


Gene symbols are in italics, except for the
symbol “+” = normal (nonmutant or wild
-
type).

Chapter 4


Genotype and Phenotype


Genotype = genetic constitution


Phenotype = observable characteristics


Brown genotype is b/b


Black mice = B/B or B/b since it only takes one
dominant black gene


Partial, or incomplete, dominance often
produces functional anomalies such as birth
defects.


Mutations that result in genotype and phenotype
changes are rare events.

Chapter 4


Homozygous and Heterozygous


Homozygote = when both genes of a pair are
the same for that gene (homozygous)



Heterozygote = genes at the same locus on a
are different for that gene (heterozygous)



Chapter 4


One Gene: Many Flavors


Ploidy

-

the number of copies of each
chromosome in a cell


Diploid
: two copies (animals consist largely of diploid cells)


Haploid
: one copy (sperm and eggs are haploid)


Plants often have three, four, or even more copies


Locus

-

the specific location of a gene on a
chromosome


Alleles

-

different forms of the same gene at a
given locus


Within a species, there may be dozens of alleles for a given
gene
.
Thus, an animal often has two different forms (alleles) of
the same gene, one inherited from each parent.

Chapter 4


DNA


deoxyribonucleic acid



A chemical structure containing the ‘blueprint’ for the
organism


Shaped like a twisted ladder, called a
double helix


Contained within the nucleus of the cell



Passed to the next generation in sperm and ova (the
gametes
)



Subject to changes known as
mutations
, produced
naturally or experimentally


Chapter 4



Single genes may affect more than one trait.


Conversely, many genes may influence the
expression of a single trait such as hair growth
(or lack of; note the nude mouse) and color.

Gene Expression

Chapter 4


Gene Inheritance

B/b

B/b


b/b

b/b


b

b

B

b

parental mating: Bb X bb


I
n the Punnett square at right, a
mating is represented by the male
genotype (Bb) on the left, crossed with
the female genotype (bb) on the top.
The alleles each parent can have in its
gametes are listed, so the male has B
and b, while the female has only b.
The possible offspring genotypes (Bb
and bb) are in the square.

For simplicity, genes are usually treated as if they
come in only two forms, or alleles, designated by a
capital letter (dominant allele), and a lower
-
case letter
(recessive allele). To show all possible ways that
offspring can inherit an allele from each parent, a
diagram, called a
Punnett square
, is used.

Chapter 4



The probability that offspring will be homozygous or heterozygous
for a given gene depends on the genotype of their parents. If both
parents are homozygous at a given locus, all offspring will be
identical
at that locus
, as shown in the following Punnett squares.

Gene Inheritance
:
a matter of chance


B
B
b
b/B
b/B
b
b/B
b/B
B
B
B
B/B
B/B
B
B/B
B/B
Chapter 4



The probability that offspring will be homozygous or heterozygous
for a given gene depends on the genotype of their parents. If both
parents are homozygous at a given locus, all offspring will be
identical
at that locus
, as shown in the following Punnett squares.

Gene Inheritance
:
a matter of chance


B
B
b
b/B
b/B
b
b/B
b/B

If either parent is heterozygous, the
probability

that offspring will
inherit different genotypes will vary, although any two individual
offspring may still be identical. For example, in the left
-
hand
Punnett square below, on average, half the offspring will be B/B.

B
b
B
B/B
B/b
B
B/B
B/b
B
b
b
b/B
b/b
b
b/B
b/b
B
b
B
B/B
B/b
b
b/B
b/b
B
B
B
B/B
B/B
B
B/B
B/B
Chapter 4


Homozygous recessive albino (AABBcc)

Dominant agouti (AaBBCc)

Homozygous recessive brown (aabbCc)

Dominant black (aaBBCc)

Putting it all together

The phenotype of coat color is determined by three
genes, each having two alleles
-

A and a, B and b,
and C and c. Different combinations of alleles
result in different coat colors.

Chapter 4



Genes on the same chromosome are physically
linked to each other and are usually inherited
together.


Consider the athymic and nude mouse.


Genes on the same chromosome are
sometimes inherited separately, due to
“crossing over” between pairs of chromosomes.


Crossing over involves chromosome breakage and rejoining.


Genes located on different chromosomes are
not linked, and are usually inherited separately.

Gene Linkage

Chapter 4


Strain and Stock Nomenclature


Inbred strains are usually designated by capital letters or a
combination of capital letters and numbers.


Substrain = line number and/or name of the person or the
laboratory developing the substrain.


The substrain symbol is separated from it by a diagonal.


A/J indicates the A strain of mouse bred by Jackson Lab.


BALB/c exception; c in this name = gene symbol for albino
.


Inbred = brother x sister (or parent x offspring) for
>
20.


Outbred stocks designated by capital letters +/or numbers.


The breeder of an outbred stock precedes the stock name
and is separated from it by a :

Chapter 4



The female’s reproductive system goes through
an estrous cycle; each cycle has four stages.

Reproduction and Breeding


Proestrus


Estrus


Metestrus


D
iestrus


Anestrus



the long period of time between
breeding seasons


Ovulation



when eggs or ova (singular is
ovum) are released from ovaries

at left, proestrus:
note the vagina of a
mouse being open,
red, and swollen

at right, not in
estrous

(Images courtesy of Angela Trupo and Dr. Kevin Barton)

Chapter 4



Sex hormones are produced
naturally in both males and
females as they mature and
influence many ‘reproductive’
traits including some anatomical
features.


descent of testes


development of mammary glands


mating behavior


Sex hormones, known as
gonadotropins,

can be
injected into females.


mimic or interrupt or synchronize
natural production


cause superovulation


Superovulation

Chapter 4


Superovulation (cont.)


Induction of ovulation can be
accomplished by IP injection of
reproductive hormones.


FSH or follicle stimulating hormone
prepares the reproductive tract for
pregnancy.


LH or leutinizing hormone causes the
release of eggs from the ovaries.


Treatment regimen varies with species.



In mice, LH is given 46 to 48 hours
after FSH.


Hormone treatment often results
in
superovulation
, an enhanced
release of ova from the ovaries.


Technique used to collect many eggs
from the same female.


Chapter 4



Gestation period


Time from fertilization to birth or parturition


Known also as pregnancy


Gestation period is specific to each species


Can vary between strains


Pseudopregnancy


Female mates with a sterile male (possibly vasectomized);
fertilization does not occur.


Act of copulation stimulates female to release hormones in
preparation to become pregnant.


Females show signs of pregnancy, including release of ova, but no
embryos result since there are no sperm and thus no offspring can
be produced.


The pseudopregnancy is brief since the unfertilized ova don’t
implant in the uterus (in mice up to 14 days of typical 21 days).

Gestation

Chapter 4



Collection of sperm or eggs/embryos


Necessary for production of some genetically
engineered mice


Important for rederivation to eliminate certain
diseases from a colony


Technique requires precise timing based on
knowledge of reproductive cycles

Artificial Insemination and In Vitro Fertilization





Chapter 4



Removal of early stage embryos up to a few days old
from the reproductive tract yields embryos for DNA
injection or freezing (cryo
-
preservation).


Taking later stage embryos, as pictured, enables study
of development and when it goes awry.


Performed surgically (for survival) and non
-
surgically
(mice are euthanized).


Survival (large animals)


Non
-
survival (rodents)


Oocytes can also be collected from females that have
not been mated (from the ovary or oviduct).

Egg and Embryo Collection

Chapter 4



Can identify stages of the estrous cycle by examining
cells taken from the vaginal wall.


Samples are collected through scraping or washing.


The stages of the estrous cycle are characterized by the
presence of cell type and condition.


Based upon the stage, timed
-
pregnant matings can be
established.

Vaginal Cytology

Chapter 4



Several factors influence which breeding system
should be used, whether…


general production of offspring is wanted (
stock
)


needing to know who the parents are (e.g.,
sire

and
dam
)


conducting test matings for sterility or
stud

performance


Monogamous and polygamous mating types are
both commonly used.


Monogamous

-

One female breeds with one male, thus it is a
breeding pair.


Polygamous

-

Two or more females breed with one male. If
1:2, then it’s a breeding trio. Poly means many; three or more
females is often called
harem

mating.


Mating Systems

Chapter 4



Intensive breeding method requires the male
and female(s) to remain together continuously.

Intensive and Nonintensive Breeding


Continuous pair or trio mating systems help
avoid fighting in some mice strains.


Whitten Effect


Presence of only females
-

no males in the colony;
may depress the estrous cycle.


Addition of male (his
pheromones
) initiates estrus
in about three days.

Chapter 4



Foster mothers are provided to young animals if
the natural mother has died, can’t nurse or
mother well, or is weakened during parturition
(dystocia).


Success is improved when offspring are close in
age to that of the foster mother’s own babies.


Some species are impossible to foster (e.g.
hamsters).


Anticipate the need for a foster mother, so set
up a coincidental mating from the ‘foster’ colony.

Foster Care

Chapter 4


Foster Care (cont.)


Healthy newborn pups
such as these will not
require fostering.


nice pink skin color


presence of milk spot


signs that mothering is
caring for them, licking
and carrying


good nest has been built

Chapter 4



Inbred strain breeding can produce animals with
unique characteristics not normally observed.


Normally recessive genes can be expressed.


Useful in research to learn the function of genes.


Sometimes embryonically lethal genes are expressed.


Having genetically identical animals is useful.


In tissue transplant studies, differing genes could result in rejection.


To minimize experimental variation.


Breeding Schemes


Foundation colony


Colony of original animals is created
or obtained


Bred to expand the colony


Resulting offspring in the production
colony are used in research projects

Chapter 4



Selective system; parents are of different inbred strains.


Offspring are thus a combination or
hybrid

of the genes
given by the parents.


Hybrid strain name is a shorthand abbreviation derived
from the two parental strains.

Hybrid Breeding

EXAMPLE:

The hybrid C3D2F1 is a first generation
(F1) cross between a C3H/He (C3)
female and a DBA/2 male (D2)


F1 offspring are identical (heterozygous for the same two
alleles at every locus), but F2 offspring, from an F1 x F1
cross, are not.

Chapter 4



Recombinant inbred strains occur


when crossing two different inbred
strains, followed by brother/sister
matings, or


when inbreeding the F1 and
subsequent generations of offspring.


Helpful in genetic assessments


Determining the inheritance of traits


Interaction (linkage) between genes



Recombinant Inbred Strains

Chapter 4



Co
-
isogenic

animals are ideal for studying
effects of one single manipulated gene while all
other genes remain identical.



Congenic

strains are used to determine how
the genetic make
-
up of an individual influences
the expression of a single gene.

Co
-
isogenic and Congenic Breeding

Chapter 4



Several factors can influence breeding:


Animal health


Of primary importance


Environmental conditions


Light, temperature, humidity, etc.


Cannibalism and desertion


Caused by inexperienced females,
overcrowding, poor environmental conditions,
stress and disturbance

Other Breeding Aspects

Chapter 4



Caging and housing arrangements


Stud male colony


Pheromones


Male and female hierarchies


Methods to verify breeding


Copulatory plug in rodents


Is not confirmation of pregnancy,only that mating has
occurred


Determine optimal breeding periods


Vaginal cytology


Proestrus, estrus, or metestrus stage


Physical and behavioral signs


Lordosis

Other Breeding Aspects (cont.)

Chapter 4



Litter size based on several factors:


age of parents; older females may suffer dystocia


nutritional status


whether an outbred or inbred strain


genetic make
-
up; some genes are embryonically
lethal in the homozygous state, so those embryos die
in utero


Some animals (e.g., mice, rats, and guinea
pigs) have a post
-
partum estrus that occurs
within 24 hours after giving birth, so re
-
mating
can occur almost immediately.

Other Breeding Aspects (cont.)

Chapter 4


Other Breeding Aspects (cont.)


Dystocia

is difficulty with birthing.


Occasionally observed in many laboratory
animal species.


A breach is an example of dystocia.


Occurs in older female guinea pigs which
have not yet had a litter because the birth
canal is smaller from fused pubis bones.


May be facilitated with oxytocin, a drug
injected to stimulate labor.


Chapter 4



Is the science of manipulating genes (DNA),
and is used to artificially alter the genetic make
-
up of living organisms to study gene function.


Mice are most often used in genetic engineering
studies; sea urchins, rats, rabbits, and sheep, too.

Genetic Engineering

at left


green fluorescent protein
(GFP) transferred from jellyfish
DNA, as seen in mouse brain
tissue



at right


a technician uses a
mouth pipette to sort mouse
embryos in preparation to
inject modified DNA

Chapter 4


Genetic Alterations


Transgenic mice


DNA from other sources (other animals,
bacteria, chemically synthesized, plants) is
inserted into the genome, at random.


Knockout mice


Blockage of function or actual removal of
specific genes on the chromosome; it is a
targeted mutation of the DNA.


Chapter 4



Three primary methods are used
to insert DNA into fertilized eggs:


Pronuclear Injection


DNA is injected directly into the fertilized
egg.


Retroviral Insertion


DNA is attached to a virus, which
carries the DNA into the egg.


Embryonic Stem Cell Insertion


DNA is purified, then inserted into
special cells via a tissue culture process
called electroporation; these cells are
then transferred into the embryos, which
are then implanted into a recipient
female.


Genetic Engineering (cont.)

Above, a chimeric
mouse, resulting from
an embryo of one
strain injected with
stem cells from
another strain; note
variations in hair color

Chapter 4



Most cells reproduce by mitosis: an identical
copy of the genome is produced, and the cell
splits into two identical “daughter” cells or
clones.


The term “clone” is also used to denote an
offspring that is genetically identical to its
parent, usually created by removing the nucleus
from an egg and inserting the nucleus from one
of the parent’s cells.

Genetic Engineering (cont.)

Chapter 4



Learn as much as you can about the genetically
engineered animals under your care.


The cost (and often luck) to produce genetically
engineered animals is enormous.


Loss of animals resulting from disease or poor
husbandry, or inaccuracies resulting from
incorrect records or improper breeding, can be
disastrous to the investigator.

Genetic Engineering (cont.)