Transgenic animals and knockout animals

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Presentation transcript:

Transgenic animals and knockout animals

3 main ways to do biological research: Do research in test tubes. Do research with cells. Do research directly with animals.

Transgenic animals and knockout animals Part 1: Transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animals? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animals.

Transgenic Animal Animal has one or more foreign genes inserted into chromosome DNA inside its cells artificially. After injecting foreign gene into the pronucleus of a fertilized egg or blastocyst, foreign gene is inserted in a random fashion into chromosome DNA: Randomly (Foreign gene may disrupt an endogenous gene important for normal development, and the chance is about 10%. ) multiple copies

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animal.

ES cell transformation Injection of gene into fertilized egg

Method 1: ES cell transformation vs Method 1: ES cell transformation vs. Method 2: Injection of gene into fertilized egg 1. ES cell transformation works well in mice only. Other transgenic animals are produced by egg injection 2. ES cell transformation provides more control of the integration step (selection of stably transfected ES cells) 3. Injection of gene into fertilized egg is less reliable (viability of eggs, frequency of integration), but it helps to avoids chimeric animals

Injecting fertilized eggs The eggs are harvested from mice (superovulated or natural matings). The DNA is usually injected into the male pronucleus. The eggs can be transferred in the same day (1 cell) or the next day (2-cells) into pseudopregnant female oviducts.

Breeding Transgenic animals (transgenic founders) Transgenic animals Individually are backcrossed to non-transgenic animals. DO NOT intercross different founders. Each founder results from a separate RANDOM transgene integration event.

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animals? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animal.

Conditional Transgenic mouse The expression of transgene in transgenic mouse can be induced

Important Considerations for Conditional Transgenes Transgenes have low or no expression when not induced Large difference between induced and non-induced gene expression Transgene expression rapidly turns on or off. Inducer (doxycycline, tamoxifen, cre) is not toxic and easily administered

Tetracycline Controlled Transactivator tTA “Tet-off” VP16 Doxycycline blocks tTA DNA binding tTA binds to tetO to activate transcription

Reverse Tetracycline Controlled Transactivator tTA “Tet-on” rtetR VP16 Doxycycline allows rtTA to bind to tetO Without doxcycline rtTA can not bind to tetO

Tetracycline Regulation: Summary No Doxycycline Doxycycline tTA expressed not expressed rtTA not expressed expressed

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animal.

Applications of Transgenic Animals Transgenic mice are often generated to 1. characterize the ability of a promoter to direct tissue-specific gene expression e.g. a promoter can be attached to a reporter gene such as LacZ or GFP 2. examine the effects of overexpressing and misexpressing endogenous or foreign genes at specific times and locations in the animals 3 Study gene function Many human diseases can be modeled by introducing the same mutation into the mouse. Intact animal provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing. 4. Drug testing

Example 1: Transgenic Cattle Cloned transgenic cattle produce milk with higher levels of beta-caein and k-casein Published in Nature, Jan, 2003

Example 2: Transgenic Mouse The growth hormone gene has been engineered to be expressed at high levels in animals. The result: BIG ANIMALS Mice fed with heavy metals are 2-3 times larger Metallothionein promoter regulated by heavy metals

Example 3: Transgenic Mouse Trangenic mouse embryo in which the promoter for a gene expressed in neuronal progenitors (neurogenin 1) drives expression of a beta-galactosidase reporter gene. Neural structures expressing the reporter transgene are dark blue-green.

Example 4: GFP transgenic mouse (Nagy) 9.5 day embryos - GFP and wt Tail tip

GFP transgenic mouse (Nagy)

Example 5: Wild and domestic trout respond differently to overproduction of growth hormone. So, GH is not effective to domestic trout.

Example 6: Transgenic mice as tools Normal mice can't be infected with polio virus. They lack the cell-surface Polio virus receptor. But, human has Polio virus receptor. Transgenic mice expressing the human gene for the Polio receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animals.

knock-out Animal One endogenous gene in an animal is changed. The gene can not be expressed and loses its functions. DNA is introduced first into embryonic stem (ES) cells. ES cells that have undergone homologous recombination are identified. ES cells are injected into a 4 day old mouse embryo: a blastocyst. Knockout animal is derived from the blastocyst.

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animals.

Vector design Recombinant DNA methods: Simple KO Structural gene desired (e.g. insulin gene) to be "knocked out" is replaced partly or completely by a positive selection marker to knock out the gene functions. Vector DNA to enable the molecules to be inserted into host DNA molecules

KNOCKOUT MICE Isolate gene X and insert it into vector. Inactivate the gene by inserting a marker gene that make cell resistant to antibiotic (e.g. Neomycin) Normal (+) gene X Genome Defective (-) Gene X Transfer vector with (-) gene X into ES cells (embryonic stem cells) VECTOR MARKER GENE e.g.(NeoR)

Vector and genome will recombine via homologous sequences Genomic gene Exon 1 Exon 2 Exon 3 Exon 4 Homologous recombination and gene disrution Grow ES cells in antibiotic containing media; Only cell with marker gene (without normal target gene) will survive

Problems with homologous recombination Unwanted random non-homologous recombination is very frequent. This method provides no selection against it Solution: Replacement vectors The knock-out construct contains the 1) NeoR gene flanked by 2) two segments of the target gene and 3) the HSVtk gene Part of the gene replaced with NeoR ES cells are selected for integration of NeoR and against integration of HSVtk* (NeoR+/ HSVtk-) on gancyclovir

Replacement vectors NeoR+/ HSVtk- NeoR+/ HSVtk+ NeoR HSVtk Homologous Gene segment 1 Gene segment 2 NeoR HSVtk Linearized replacement plasmid NeoR Homologous recombination NeoR+/ HSVtk- Random integration NeoR+/ HSVtk+ HSVtk will convert gancyclovir into a toxic drug and kill HSVtk+ cells

Typical KO vector *tk:thymidine kinase

Inject ES cells with (-) gene X into early mouse embryo Transfer embryos to surrogate mothers Resulting chimaras have some cells with (+) gene X and (-) gene X. Mate them with normal mice It is lucky, if germline contain (-) gene X Screen pups to find -/+ and mate them Next generation will split as 3:1 (Mendelian)

Embryonic stem cells Harvested from the inner cell mass of mouse blastocysts Grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.

ES cells growing in culture

ES cells are transformed Cultured ES cells are exposed to the vector Electroporation punched holes in the walls of the ES cells Vector in solution flows into the ES cells The cells that don't die are selected for transformation using the positive selection marker Randomly inserted vectors will be killed by gancyclovir

Successfully transformed ES cells are injected into blastocysts

Implantation of blastocysts The blastocysts injected with transformed ES cells are left to rest for a couple of hours Expanded blastocysts are transferred to the uterine horn of a pseudopregnant female Max. 1/3 of transferred blastocysts will develop into healthy pups

Implanting blastocysts 1 2

Implanting blastocysts 3 4

Testing the offspring A small piece of tissue - tail or ear - is examined for the desired gene 10-20% will have it and they will be heterozygous for the gene

Breeding Chimeras (knock-out founder) Chimera - the founder germ-line transmission - usually the ES cells are derived from a 129 mouse strain (agouti or white colour) and the ES cells are injected into blastocyst derived from a C57Bl/6 mouse (black). The more that the ES cells contribute to the genome of the knockout mouse, the more the coat colour will be agouti. The chimera mouse is usually “tiger” striped.

Breeding Chimeras (knock-out founder) Males that are 40% to 100% based on agouti coat colour should be bred Females should not be bred (low incidence of success). Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting knockout gene, the knockout gene will not likely go to germline and should not be used for more breeding.

Littermates Black mouse - no apparent ES cell contribution Chimeric founder - strong ES cell contribution Chimeric founder - weaker ES cell contribution

Chimeric mouse

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animal.

Conditional knock-out animals How to make FLOXed gene Gene of interest NeoR TK Electroporate targeting vector into ES cells, followed by +/- selection loxP loxP NeoR+/ HSVtk- cells selected loxP loxP Gene flanked by loxP sites (floxed) Make mice and breed floxed allele to homozygousity.

Mate FLOXed mice with mice carrying a Cre transgene Marker gene Promoter elements Cre IRES GFP SV40 p(A) intron Crucial element. Recombinase would be expressed in accordance with specificity of your promoter. Promoter could be regulated !!! artificailly or naturally

Conditional knock-out animals inactivate a gene only in specific tissues and at certain times during development and life. Your gene of interest is flanked by 34 bp loxP sites (floxed). If CRE recombinase expressed Gene between loxP sites is removed

Transgenic animals and knockout animals Part 1: transgenic animals: Introduction to transgenic animals. How to make transgenic animals? How to make conditional transgenic animal? Applications of transgenic animals. Part 2: Knockout animals Introduction to knockout animals. How to make knockout animals? How to make conditional knockout animals? Applications of knockout animal.

Applications of Knock-out animals Find out if the gene is indispensable (suprisingly many are not!) Check the phenotypes of knockout animals Determine the functions of knockout gene.

Health Monitoring Programs Costly Monitor health status of colony Long-term savings: time, effort, money Inform investigator (collaborators) of pathogen status Prevent entry of pathogens Promptly detect and deal/eliminate pathogen entry

Health Monitoring Programs Months of research data may have to be thrown out because of undetected infection: Unfit for research Data unreliable

Pathogens Viral, bacterial, parasitic, and fungal Sometimes no overt signs Many alter host physiology - host unsuitable for many experimental uses Cures can be bad too!

Pathogens: Some common pathogens and their effects Sendai virus Mouse, rat, hamsters One of the most important mouse pathogens Transmission - contact, aerosol - very contagious Clinical signs - generally asymptomatic; minor effects on reproduction and growth of pups

Pathogens (cont): Some common pathogens and their effects Infected shortly after birth stop breeding Altered physiology: as the virus travels down the respiratory tract -necrosis of airway epithelium, pneumonia in lungs, lesions. 129/J and DBA, aged and immunodeficient mice most susceptible; SJL/J and C57Bl/6 most resistant

Pathogens (cont): Some common pathogens and their effects Reported effects Interference with early embryonic development and fetal growth Alterations of macrophage, natural killer (NK) cell, and T- and B-cell function Pulmonary hypersensitivity Wound healing

Pathogens (cont): Some common pathogens and their effects MHV Probably most important pathogen of laboratory mice Extremely contagious; aerosol, direct contact; No carrier state Clinic state: varies dependent upon MHV and mouse strains

Pathogens (cont.): Some common pathogens and their effects Diarrhea, poor growth, death Immunodeficient (e.g. nu/nu) wasting syndrome -eventual death Reported effects: necrotic changes in several organs, including liver, lungs, spleen, intestine, brain, lymph nodes, and bone marrow; differentiation of cells bearing T-lymphocyte markers; altered enzyme activities, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc.

Pathogens (cont.): Some common pathogens and their effects Helicobacter spp H. Hepaticus (mice) most prominent Transmission: direct fecal-oral Clinical signs absent in immunocompetent mice In immunodeficient mice- rectal prolapse Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms

Pathogens (cont.): Some common pathogens and their effects Oxyuriasis (Pinworms) Mouse pinworms (Syphacia obvelata) has been reported to infect humans Eggs excreted in faeces, can aerosolize - wide spread environmental contamination Infection rate high; infection usually sub clinical Athymic (nu/nu) mice are more susceptible

Pathogens (cont.): Some common pathogens and their effects Few reports documenting the effects of pinworms on research, many consider irrelevant Acariasis (mites) Hairless mice not susceptible Transmission - direct contact Eradication very labour-intensive

Pathogens (cont.): Some common pathogens and their effects Reported to have caused: altered behaviour selective increases in immunoglobulin G1 (IgG1), IgE, and IgA levels and depletion in IgM and IgG3 levels in serum Lymphocytopenia Granulocytosis Increased production of IL-4; decreased production of IL-2

The End and Good bye!