Breeding & Genetic Engineering. Laura Garzel, DVM

Breeding & Genetic Engineering Laura Garzel, DVM

Part I GENETICS

Genetics Definition: the biological science dealing with heredity Gain an understanding of why offspring have similarities and differences from their parents Inherit characteristics from parents Differences caused by environment or genetic factors

Genes Traits: physical characteristics Fur color, eye color, height, number of toes Genes: units of inheritance it Traits are passed from one generation to the next as genes. Every characteristic of an organism is determined by the genes it received from its parents.

Chromosomes Genes are located on chromosomes Chromosomes: structures made up of deoxyribonucleic acid, or DNA Paired Located in cell nuclei

Chromosomes Sperm and ova have unpaired chromosomes When the female s ovum is fertilized by the male s sperm, the resulting zygote has paired chromosomes Half of each parent s genes are passed to the next generation

Alleles Most organisms have two sets of chromosomes, one set inherited from each parent Genes at the same location on the two matching chromosomes are called alleles Alleles for a gene can be dominant Easily expressed, only 1 copy needed Alleles for a gene can be recessive Less likely to be expressed, 2 identical copies needed

Alleles Locus: a location on a chromosome Homologous: same / matching pair

Alleles A species characteristic is commonly more complex than the simple dominant/recessive allele relationship More than two alleles can be responsible for a trait

Gene Symbols Capital letters: represent dominant alleles Lower case letters: recessive alleles + symbol: represents wild-type, or normal (nonmutant) gene Wild-type mouse Gene symbol: + Obese mouse Gene symbol: ob

Homozygotes & Heterozygotes Zygote: the cell produced at fertilization by the union of a male gamete (sperm) and a female gamete (egg) Homozygote: when both alleles of a gene are the same Only have one type of gamete to pass on to the next generation Heterozygote: when the 2 alleles of a gene are different Have 2 types of gametes to pass on to the next generation

Genotype & Phenotype Genotype The genetic makeup of an animal Phenotype The physical characteristics of an animal Genotype: wh/wh Phenotype: white fur

Genotype & Phenotype Are these tiger cubs homozygous or heterozygous for the white gene? Is the white gene dominant or recessive? Genotype: wh/wh Phenotype: white fur

Genotype & Phenotype Are these tiger cubs homozygous or heterozygous for the white gene? Homozygous: both alleles are the same Is the white gene dominant or recessive? Recessive: lower case letters Genotype: wh/wh Phenotype: white fur

Gene Expression x bb Recessive brown coat (two recessive alleles) BB Dominant black coat (two dominant alleles) What color will their puppies be??

Gene Expression x bb Recessive brown coat (two recessive alleles) B B BB Dominant black coat (two dominant alleles) Punnett Square b Bb Black Bb Black b Bb Black Bb Black

Gene Expression x bb Recessive brown coat (two recessive alleles) BB Dominant black coat (two dominant alleles) Bb Black puppies!

Gene Expression x bb Bb Recessive blue eyes Dominant brown eyes (two recessive blue alleles) (one dominant brown allele, one recessive blue allele) What color eyes will their kittens have??

Gene Expression x bb B b Bb Punnett Square b Bb Brown bb Blue b Bb Brown bb Blue

Gene Expression x bb Bb bb Bb Half with blue eyes, half with brown eyes!

Gene Expression Pleiotropic effect: a single gene may affect more than one trait Polygenic effect: many genes may influence the expression of a single trait

Mutations Mutation: change in a gene Can be spontaneous Can be caused by chemical or environmental factors Can be harmful, beneficial, or benign Scottish fold cat

Gene Linkage Linked genes Next to one another on the same chromosome Typically y inherited together Genes located on different chromosomes are usually inherited separately S t t i bi ti diff t f th t f Segregate, or reassort, in combinations different from that of either parent

Genetic Engineering The science of manipulating the genetic make- up of a living organism Variety of species Bacteria, viruses, plants, animals Mouse most often used in genetic engineering Two types of genetically engineered mice Transgenic mice Knockout mice

Transgenic Mice DNA from a different animal is inserted into their genes The inserted gene g functions as it would in the donor animal

Transgenic Mice First step: making the DNA that will be inserted Several different components attached together Code for a protein of interest Determine where, when, and how much protein is made Done in a test tube Left: mouse with growth hormone gene inserted next to another gene produced at high frequency: large amount of growth hormone produced Right: wild-type mouse

Creation of Transgenics Next step: pronuclear injection DNA injected directly into the fertilized egg (pronucleus) with a tiny glass pipette Zero, one, or multiple copies of DNA may integrate randomly into the pronuclear chromosomes Egg then transplanted into a female for gestation

Stem Cells Capable of becoming any of the different cells that make up the various body tissues (pluripotent) Cell mass inside of the developing zygote

Gene-targeted Mutations The genetic change is purposely targeted to a specific gene DNA is inserted into stem cells Wild-type gene replaced by the new gene

Gene-targeted Mutations These stem cells (donor cells) injected into an embryo (recipient cells) Half the embryo is the donor, half is recipient Embryo implanted into female + = Donor cells: stem cells containing gene of interest (brown mouse background) Recipient cells: early stage embryo with its own stem cells (white mouse) Final embryo: donor + recipient stem cells Surrogate mother

Gene-targeted Mutations The resulting offspring animal is a chimera Half donor cells, half recipient cells An animal with about half of its tissues containing the desired DNA Donor cells (with gene of interest): brown mouse Recipient cells: white mouse

Gene-targeted Mutations Germ-line transmission The gene of interest may or may not be in the chimeria s germ cells Breed the chimera to wild-type mice to determine if their offspring carry the gene

Gene-targeted Mutations Donor cells (with gene of interest): brown mouse Recipient cells: white mouse Brown offspring Carry gene of interest White offspring Carry the wild-type gene

Genotyping Tissue samples from young animals are taken for genetic analysis Typically tail clips Determine if the gene or mutation of interest is present in the animal Based on the results, breeding pairs are established to expand the colony

Knockout Mice Targeted mutations Two possibilities: The function of a gene is blocked The specific gene is removed from the animal s genetic composition Gene is knocked out Useful model of genetic disease Determine the effect of a gene when it is missing or abnormal

Contribution of Genetic Engineering Understanding of the role genes play in: Human and animal biology and health Disease processes The knowledge gained could improve and extend the lives of all humans.

Part II REPRODUCTION & BREEDING

Reproduction Gamete production Sperm produced in male testes Eggs, or oocytes, produced in female ovaries Gestation period The time required for a zygote to develop into a fetus and be born Gestation length varies by species Parturition Giving birth

Reproduction Fertilization Location and manner in which h the eggs and sperm come together depends on the species Most mammals: fertilization within female s reproductive tract Amphibians: outside tid the bd body

Reproduction Development of fertilized egg Also depends d on species Mammals: grows in female s uterus Birds and reptiles: outside the body but in an egg

Reproduction Estrous cycles Female s ova undergo changes to prepare them for fertilization Length and frequency of estrous cycles depend on species Monoestrous: one cycle per year Polyestrous: repeated cycles in a year Bears have their breeding season in the spring to allow their cub to grow during the warm seasons

Reproduction Estrous cycles Four stages Proestrus Estrus Metestrus Diestrus

Estrous Cycle Proestrus Eggs develop in the follicles of the ovary Female begins to react sexually toward males Egg Follicle Ovary

Estrous Cycle Estrus Maximum receptivity of female to mating Egg enters oviduct Oviduct Egg

Estrous Cycle Metestrus and diestrus Female ignores male Nonpregnant female: reproductive tract reverts to the original state Anestrus The long period between breeding seasons in some species Dogs, cats

Estrous Cycle: Dogs External signs observed during the estrous cycle Proestrus: restless, increased appetite, swollen vulva with bloody discharge Estrus: highly restless, less blood, swollen vulva, accepts male Metestrus: vulva becomes less swollen female Metestrus: vulva becomes less swollen, female fights off male, assumes normal behavior

Vaginal Cytology Helps to determine the stage of the estrous cycle Specific types and quantities of cells observed Useful for establishing timed-pregnant matings Rodents, dogs, NHPs Cells collected by a wash or by swabbing Transferred to a microscope slide

Vaginal Cytology Proestrus: small, round epithelial (squamous) cells Estrus: irregularly-shaped (cornified) squamous cells Diestrus: leukocytes and round epithelial cells Metestrus: cornified cells and leukocytes (white blood cells)

Superovulation Induced ovulation Increased number of eggs that mature and are released from the ovary at the same time Animal injected with a combination of gonadotropins (reproductive hormones) Used when eggs are collected from the female Useful for collecting embryos to use in the creation of transgenic mice

AI & IVF AI: Artificial insemination Female superovulated Semen collected from male Semen manually placed into reproductive tract of female IVF: In vitro fertilization Union of eggs and sperm outside the body Resulting zygote implanted into a pseudopregnant female where it develops

Egg & Embryo Collection Egg collection Rodents: female superovulated, euthanized, eggs flushed from oviducts Large animals: survival surgery to aspirate eggs from ovaries or flush from oviducts Embryo collection After natural fertilization, before implantation Transferred to another female Why? Production of transgenic and knock-out animals Rederivation to eliminate disease

Breeding Schemes Inbreeding To produce animals with minimal genetic variation These closely related animals are identified as members of a strain Inbred after 20 generations of brother x sister or parent x offspring breedings

Breeding Schemes Outbreeding Animals are of the same stock Unrelated animals of the same stock are mated Results in maximum amount of genetic difference

Breeding Schemes Line breeding Animals with a similar characteristic that share a common ancestor are mated

Breeding Schemes Cross breeding Mating of animals of different breeds

Breeding Schemes Hybrid breeding Male and female are of different inbred strains Offspring is a mixture (hybrid) of its parents First generation of hybrids is phenotypically and genotypically identical Used to transfer a desired mutation to another strain Designation: two parental strains, female parent first C3D2F1 = first generation (F1) cross between C3H/He female and DBA/2 male

Breeding Schemes Recombinant inbred strains Two different inbred strains crossed Then brother/sister matings Help to determine inheritance of traits that are influenced by several genes Help to identify linkage between genes

Breeding Schemes Recombinant inbred strains

Breeding Schemes Coisogenic breeding An animal of an inbred strain that has a spontaneous mutation = coisogenic animal Coisogenic animal bred to members of the same strain to maintain the mutation The two strains differ by only one gene Used to study the effects of one gene when all other Used to study the effects of one gene when all other genes remain identical

Breeding Schemes Coisogenic breeding x

Breeding Schemes Congenic breeding An animal having a mutation of interest is mated to an inbred animal from a strain of choice After continued matings with that strain, the mutation becomes fixed Help determine how the genetic make-up of an individual influences the expression of a single gene

Breeding Schemes Congenic breeding x x x x

Strains and Stocks Nomenclature Inbred strains Capital letters or combination of capital letters and numbers DBA, C57BL Substrain symbol: number of the line, or name of the person or lab who developed substrain A/J (J = Jackson Labs) Outbred stocks Capital letters or combination of capital letters and numbers Breeder of the stock precedes name Crl:SW

Mating Systems Monogamous mating One male and one female selected by the breeder Harem mating AKA polygamous mating system One male with two or more females Most young from least breeders Separate housing Brought together only for breeding Best when males aggressive towards the young

Intensive Breeding Intensive breeding method Male with female(s) continuously Multiple males if parentage unimportant Whitten effect Adding a male to a cage of females causes all females to go into estrus in about three days

Nonintensive Breeding Male and female housed separately while female is pregnant Female mates again when young are weaned

Foster Care Foster mother: lactating animal that raises the young of another female If mother dies, is sick, cannot provide enough milk, or fails to care for her babies Best with young animals, close to the age of the foster mother s offspring

Foster Care Can be done to create disease free young Young delivered by sterile surgery (cesarean section) and given to pathogen-free foster mothers Possible because most pathogens are not transferred across the placenta Some species may be impossible to foster May cannibalize the young of a different mother Hamsters

Dystocia Dystocia Difficult birth Can be minimized in guinea pg pigs if the female is bred before they reach six months of age After six months the pubic bone fuses, narrowing the birth canal

Dystocia

Special Considerations Animal health is of primary importance in all species used for production It is a poor management practice to establish colonies with foundation animals with disease Environmental considerations Correct light cycle, temp, humidity are vital

Special Considerations Remember species differences Some species housed together only when female is in estrus Female brought to male s cage to prevent female from needing to defend her territory Female returned to her own cage after mating Nonhuman primates, rabbits, hamsters

Special Considerations Special care of breeding animals Quiet area away from busy parts of the facility Appropriate p caging g to accommodate parturition and numerous offspring Nesting boxes for appropriate species

Special Considerations Post-partum estrus Estrus within 24 hours after giving birth Mice, rats, guinea pg pigs Remain pregnant while nursing previous litter First litters Smaller than later litters Mothers give poorer care: higher mortality rate

Special Considerations It is wise to delay husbandry practices which may not be immediately necessary for a few days when dealing with young animals and their mothers Some animals (rabbits and rodents) may desert, kill, or cannibalize their young if disturbed Minimal handling of newborns is a general rule

Animal Identification Important for efficient operation of breeding systems Cage cards Animal s history and genetic background, types of experiments, PI contact information, protocol Short-term term individual identification Fur clipped in various places, patterns Nontoxic dyes, markers on fur or tail Collars Cats, dogs, nonhuman primates USDA number for cats and dogs

Animal Identification Permanent Identification Ear notching: predetermined code, easy to read, little trauma to ear

Animal Identification Permanent identification Toe clipping: first bone of certain toes removed Rodents Discouraged unless strong justification provided to IACUC Under anesthesia Wing tags: small clips with ID numbers Leg bands are alternative to wing tag

Animal Identification Ear tags Near the base of the ear Dangle like an ear ring Can be lost due to fighting, grooming, or infection of the site

Animal Identification Tattooing Ears, tails, or toes Chest or inner thigh for nonhuman primates Mechanical, hand-operated clamp Electrical, pen-like tool Sanitize between animals

Questions What is the term for a unit of inheritance? A. DNA B. Chromosome C. Gene D. Zygote

Questions When is a recessive gene usually expressed? A. When 2 recessive alleles are present. B. When there is 1 recessive and 1 dominant allele. C. When only 1 chromosome is present. D. When the 2 alleles are the same.

Questions Where are unpaired chromosomes found? A. In birds B. Zygote C. Only under laboratory conditions D. Ova and sperm cells

Questions What does + as a gene symbol represent? A. The mutant type B. The normal or wild type C. The dominant allele D. The unknown allele

Questions What is gene linkage? A. Genes located adjacent to one another on the same chromosome that tend to be inherited B. Genes that influence one another C. Genes that overlap and create mutations D. Genes that t express multiple l traits

Questions What is gene linkage? A. Genes located adjacent to one another on the same chromosome that tend to be inherited together B. Genes that influence one another C. Genes that overlap and create mutations D. Genes that express multiple traits

Questions The period between fertilization and birth is also known as: A. Gestation B. Reproduction C. Ovulation D. Parturition

Questions Which of the following helps to ensure successful fostering? A. Animals close in age to the original litter B. Close to the same number as the original litter C. The same coat color of the original litter D. All of the above

Questions What is an advantage to tattooing non-human primates on the chest? A. It rarely needs to be redone. B. They can be identified without anesthetizing. C. This area is hairless so there is less preparation. D. There is more space to make the numbers larger.

Questions What is the correct order? A. Genes form chromosomes that form DNA B. DNA forms genes that form chromosomes C. DNA form chromosomes that form genes D. Chromosomes form DNA that form genes

Questions What happens in the formation of a zygote? A. The unpaired chromosome makes a matching pair B. One parent's genes dominate the other parent's genes C. One half of each parent's genes are passed to the next generation D. The parent chromosomes mutate

Questions In which type of animal does fertilization occur outside the body? A. Birds B. Most amphibians C. Marsupials D. Whales

Questions What occurs as part of the estrous cycle? A. Gestation B. Reproduction C. Ovulation D. Parturition

Questions What is the most effective way to tell what stage of the estrous cycle an animal is in A. Observation for mating behavior B. Observation for physical changes C. Observation for vaginal discharge D. Analyze cell samples from the vagina

Questions What is the breeding scheme for producing animals with minimal genetic variation? A. Outbreeding B. Inbreeding C. Line breeding D. Cross breeding

Questions When is a strain considered inbred? A. Anytime as long as it isn't contaminated by a difference B. Up to 20 generations of brother-sister matings C. After a minimum of 20 generations of brother-sister matings D. When each animal has an identical set of genes

Questions When is a strain considered inbred? A. Anytime as long as it isn't contaminated by a difference B. Up to 20 generations of brother-sister matings C. After a minimum of 20 generations of brother- sister matings D. When each animal has an identical set of genes

Questions What is an advantage of monogamous mating? A. Most economical B. Maximum production C. Longer breeding life D. Simple record keeping

Questions What animals are especially prone to cannibalizing their young if disturbed? A. Rodents and rabbits with their first litter B. Animals in dirty cages C. Group housed rodents D. Sick animals

Any Questions?