Genetics and Breeding

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.

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.

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.

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).

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.

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)

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.

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

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.

parental mating: Bb X bb b b B B/b B/b b b/b b/b 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.

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.

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.

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)

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.

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 :

Reproduction and Breeding • The female’s 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

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

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.

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 don’t implant in the uterus (in mice up to 14 days of typical 21 days).

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

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).

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.

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 1:2, then it’s a breeding trio. Poly means many; three or more females is often called harem mating.

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.

Foster Care • 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 (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

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

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

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

Co-isogenic and Congenic Breeding • Co-isogenicanimals are ideal for studying effects of one single manipulated gene while all other genes remain identical. • Congenicstrains are used to determine how the genetic make-up of an individual influences the expression of a single gene.

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

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

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.

Other Breeding Aspects (cont.) • Dystociais 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.

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

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.

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

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 parent’s cells.

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.

Genetics and Breeding