Genetics and Breeding

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Genetics and Breeding

• LAT Chapter 4

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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.

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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.

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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.

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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).

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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.

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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)

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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.

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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

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Gene Expression

• 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.

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Gene Inheritance
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.

• In 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.

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Gene Inheritance a matter of chance

• 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.

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Gene Inheritance a matter of chance

• 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.

• 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.

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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.
Homozygous recessive albino (AABBcc) Dominant
agouti (AaBBCc) Homozygous recessive brown
(aabbCc) Dominant black (aaBBCc)
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Gene Linkage

• 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.

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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 gt20.
• 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

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Reproduction and Breeding

• The females reproductive system goes through an
  estrous cycle each cycle has four stages.

• Proestrus

at left, proestrus note the vagina of a mouse
being open, red, and swollen at right, not in
estrous

• Estrus

• Metestrus

• Diestrus

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

• Anestrus the long period of time between
  breeding seasons

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

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Superovulation

• 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

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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.

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Gestation

• 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 dont implant in the uterus (in
  mice up to 14 days of typical 21 days).

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Artificial Insemination and In Vitro Fertilization

• 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

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Egg and Embryo Collection

• 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).

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Vaginal Cytology

• 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.

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Mating Systems

• 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 12, then its a breeding trio. Poly
  means many three or more females is often called
  harem mating.

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Intensive and Nonintensive Breeding

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

• 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.

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Foster Care

• Foster mothers are provided to young animals if
  the natural mother has died, cant nurse or
  mother well, or is weakened during parturition
  (dystocia).
• Success is improved when offspring are close in
  age to that of the foster mothers 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.

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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

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Breeding Schemes

• 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.

• 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

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Hybrid Breeding

• 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.

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

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Recombinant Inbred Strains

• 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

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Co-isogenic and Congenic Breeding

• 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.

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Other Breeding Aspects

• 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

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Other Breeding Aspects (cont.)

• 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

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Other Breeding Aspects (cont.)

• 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.

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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.

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Genetic Engineering

• 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.

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
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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.

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Genetic Engineering (cont.)

• 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.

Above, a chimeric mouse, resulting from an embryo
of one strain injected with stem cells from
another strain note variations in hair color
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Genetic Engineering (cont.)

• 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 parents cells.

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Genetic Engineering (cont.)

• 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.