aalas lat chapter 4 genetics and breeding...
TRANSCRIPT
What is the fundamental physical unit of heredity?
GENEBACKBONE
Base Pairs
Heredity Characteristics are determined by elementary units call genes Carried on chromosomes Found in cell nuclei (image) composed of DNA (deoxyribonucleic acid)
Pass from generation to generation in a predicable manner Asexual reproduction (lower –bacteria) Sexual reproduction (higher)
Central Dogma
http://en.wikipedia.org/wiki/File:Central Dogma of Molecular Biochemistry with Enzymes.jpg
WITHIN THE CELL NUCLEUS
OUT IN THE CELL CYTOPLASMA
Think of the locus as the address, the gene as the house at that address, the allele would be the type of house (A frame, ranch, two‐story)
FROM MOM
FROM DAD
LOCUS
Allele A
Allele B
Pair of Chromosomes
Genes vs. Alleles
FVBFVB
C57BL/6 C3H
DBA
The gene for coat color has many different alleles that can be expressed
Dominance/Recessive
Gene Expression A number of different genes affect coat color Agouti ‐ A (black at the base and yellow at the tip)
a – solid color hair Black – B
b ‐ brown Albino – cc (prevents expression of color)
CC or Cc – color determined by other genes Partial/Incomplete Dominance
How to express genes A – dominant a ‐ recessive Wv – anemia and sterility wv – staggering gate Ilalics + ‐ used to represent wild type (aka normal or non mutant)
B6.Cg-Foxn1nu/JCrl:NU-Foxn1nu
Crl:CD1-Foxn1nu
Gene expression A single trait can be impacted by many genes A single gene may affect more than one trait S1 gene – Piebald lethal gene
Hair coat Abnormal GI tract
Phenotype
Zygositydef: the number of copies of an allele
Zygote: the cell produced at fertilization by the union of a male and female gamete (sperm and egg)
Homozygous ‐ 2 copies of the same gene Heterozygous – different genes on the same locus
Table 4.1Examples of Mouse Coat Color
Gene Expression
Gene linkage Linked genes are located adjacent to one another on the same chromosome and tend to be inherited together Fluit fly : Drosophila melanogaster black body/vestigial wings
Genes on different chromosomes are inherited separately Segregate and reassort
One Set of Chromosomes (Human)
Strain and Stock nomenclature Outbred stocks (capital letters)
SW and ICR : indicates holder and precedes the stock name
Crl:SW
Inbred strain (capital letters +/‐ numbers) AKR, DBA, C57BL, 129 (rare)
Substrain Number of the line and/or person or lab
A/J, C57BL/6NCrl BALB/C (C‐albino)
http://jaxmice.jax.org/support/nomenclature/tutorial.html
Other rodents Rats
Outbred stock SD – Sprague Dawley LE – long evens
Inbred strain F344 – Fisher rat LEW – Lewis rat
Guinea pigs Inbred strain
Strain 2 and 13
BALB/c StCrl p GF (F83)
Strain Symbol (All Capitals and/or numbers)
Diagonal/ separates strain from substrain
Substrain Designation
Subline Derivation
Hygienic State
Mice name BALB, with albino gene, originally inbred by strong, now by charles river from freeze preserved embryos. Mice are germ free and inbred for 83 generations
Reproduction and Breeding Estrous cycle
Four stages:
proestrus, estrus, metestrus, diestrus
Anestrus – time between cycles, length is species dependent
Monoestrus vs polyestrus vs seasonal polyestrus
Eggs (ova) develop in
the follicles of the ovary,
receptive to male
Maximal receptivity, ova enter
the oviduct, lordosis,
Non responsive, reproductive tract
returning to normal
Terminology False pregnancy/pseudopregancy
Female mates with a sterile male No fertilization Nest building and milk production, shorter
Gestation Time from fertilization to birth (parturition)
Superovulation = Inject gonadotropins (reproductive hormones) Increased number of eggs that mature and release from the ovary
Terminology Intromission (Coitus)
Male inseminating the female with sperm Artificial Insemination (AI) and in vitro fertilization
Manually placing semen into the reproductive tract Female: naturally cycling, hormone induced Male: artificial vagina, manual or electro ejaculation, or directly from the testis/epididymus
In vitro = union of eggs and sperm outside the body Zygote can be implanted into a pseudopregnant uterus or oviduct for normal development
Egg and Embryo collection
Eggs Unfertilized eggs aspirated directly or flushed
Embryo collection Fertilized eggs Prior to implantation in the uterine wall
Embryo transfer Requires careful planning and time
Dam mated with a vasectomized Sire = Pseudopregnant Why?
Sensitive mutant mice Create transgenic and targeted mutations (knockout) animals
Clean up a colony aka rederivation Only for agents that are infectious after birth (horizontal) Not for agents transferred in utero(vertical)
Vaginal Cytology Used to determine stage of estrus cycle microscopically Wash
Flush with sterile fluid, centrifuge, smear on a slide
Sterile swab inserted into the vagina and rotated Collects cells from the vaginal wall
Estrous cycle Cycles are species specific Rats
Proestrus: squamous cells (12 hours) Estrus: cornified squamous cells (9‐15 hours) Metestrus – leukocytes (WBC) + few cornified squamous (14‐18)
Diestrus – leukocytes and round epithelial cells (60‐70 hours)
http://cancerres.aacrjournals.org/content/70/1/221.full
Inflammatory cells (WBC)
Squamous epithelial cells (immature-(lt blue)
Epithelial cells, Inflammatory cells
Cornified Epithelial cells (mature-(orange)
Mating Systems Non intensive Male removed and not returned until pups are weaned
Intensive breeding Male remains with the female
Many males and females – don’t know parents, fighting One male, many females – less fighting, shared care Post partum estrus (24 hours)
Monogamous and Polygamous systems Harem breeding Gerbils: monogamous pairing
Hormone effects Group housed females = anestrus (Lee Boot)
Hormone expression results in suppression of estrus
Whitten effect Exposure to a male (or male urine) will cause females to go into estrus on day 3
Used to synchronize for timed mating
Foster care Lactating dam that cares for young that don’t belong to her
Want the young to be close in age Used to eliminate pathogens from colony
Pregnant dam‐ pups delivery via cesarean section (sterile) and given to clean dams
Can create specific litter size/gender
CAN’T CROSS-FOSTER HAMSTERS!!!
Breeding Scheme Founders
Transgenic – original animals that have incorporated the desired DNA into their cells
Founders expansion colony Helps to ensure that the desired trait will not be lost
Expansion colony production colony Use in experiments
Breeding Scheme Hybrid breeding
Cross two inbred lines Shorthand combination of the parent strains C3D2F1 ‐ C3H/H3 dam crossed with a DBA/2 male, 1st gen
Recombinant inbred Crossing two inbred lines then brother sister mating Used to determine gene linkage
Breeding Scheme Coisogenic
Animal displaying a spontaneous mutation (differs by 1 gene only), breed to the original background strain
Congenic breeding Selectively mating a mouse with a mutation to a specific strain
Selecting mice with the mutation (F1) and continue to breed to inbred strain, 10‐12 cycles to be fixed
Genetic make up of an individual influences on the expression of a single gene
http://jaxmice.jax.org/support/nomenclature/tutorial.html
Other breeding aspects Cannibalism and desertion
Inexperienced female Overcrowding Poor environmental conditions Disturbance or stress
Caging – larger, nest boxes, metal cages (cold), wire bottom
Environment Light cycle, temperature, humidity Quiet area, reduce noice improves success
Breeding info Female defends territory (take her to the male cage) Confirmation of copulation
Vaginal plug Swab and look for sperm
http://www.thefunmouse.com/info/breeding.cfm
Signs of estrus Dogs
Proestrus: restless, hungry, swollen vulva, discharge Estrus: less blood in discharge, mounting, rapid turning on the hindlegs and stamping of forelegs, acceptance
Metestrus: fights off male, return to normal, vulva less swollen
Non Human Primate Sex skin
Queen (cats) 1‐2 days prior to estrus vocalization, flattening the back and elevation of the hindquarters,
Lab animal litters First few litters small Post partum estrus (rats, mice, guinea pigs)
Dystocia Guinea pigs – mate before 6 months of age WHY?
Pelvic symphysis will ossify around 7 months if not bred Cesarean section will be required to deliver pups
Young G. pigs have a fibrocartilagenous symphysis flexible so big pups can pass out of vaginal canal (Gestation is 59‐72 days)
This term describes an animal containing genetic material artificially transferred from another species or breed? Transgenic
Genetic engineering Transgenic ‐ Why would we do such a thing?
DNA from a different animal inserted into genes Function (make human protein) Block function/remove
Targeted mutation Knockout
http://www.biochem.mpg.de/en/facilities/transgene/
How can we do such a thing? Pronuclear injection
Inject DNA into fertilized egg Retroviral insertion
Attach DNA to a virus, carries to zygote for introduction into cell
Modified virus (no DZ) Embryonic stem cell insertion
DNA inserted into a stem cell via electroporation (electric current)
http://www.biochem.mpg.de/en/facilities/transgene/
Genetic engineering In vitro (outside the animal)
Zygote then placed in a surrogate mother Only small numbers survive Only a small number of those incorporated the DNA
Chimera (see chart next page) DNA will incorporate into some but not all of the cells Hope that it is in the germ cell s(egg and sperm)
Genotype Collect a small piece of tissue and look for copies of the DNA
Chimera Types of cells from the 129 strain (injected embryonic stem cells) as well as from the B6 strain fertilized egg (zygote)
CLONING Correct disease in humans by substituting the correct gene for the one with the defective one
1st clone of an adult mammal Nucleus from a mammary gland cell of a sheep and inserted it into the nucleus of an egg cell
Placed in a donor animal Offspring= clone
100% homozygous at all loci No breeding/exact copy of the genetic material in the cell of the donor
Wilmut stated "Dolly is derived from a mammary gland cell and we couldn't think of a more impressive pair of glands than Dolly Parton's".[6]