GloFish, the first genetically modified animal to be sold as a pet

Introduction of Exogenous DNA in to the genome such that it is stably maintained in a heritable manner. The genes transferred are otherwise not possible to transfer by conventional and natural methods transfer of genetic material.

It has broken the Biological barriers of species, strains even kingdoms in plants, animals and microorganisms.
Example: Sheep producing glycosylated -1-antitrypsin @ of 35g/lt of milk, the gene having cloned in the ovine -lactoglobulin promoter Doly the sheep ? (Wilmut & Campbel) Roslin Inst. Scotland 1997) fertile, arthritis ? Clone Poly = +human clotting factor IX Rice with dafodill gene synthesise Vitamin A Bt cotton has the insert of Bollworm resistance gene from microorganism Bacillus thuringiensis Vegetables delivering vaccine against cholera and rabies

Considerations
Safe : free from herbicides, pestcides, antibiotics, infections Quality : biological activity maintained over time / season/ year/ location/ high cell density / high conc. / edible vaccine Processing: easy & cheap. Post translational & post transcriptional modifications complete, good expression levels No /minimum adverse effect in producing plant or animal Recovery from milk / urine / fruits etc. Cost:

Question: Which of the following would appropriately be described as a transgenic organism? A. The sheep Dolly, which was produced by cloning. B. A sheep that produces human a-1-antitrypsin in its milk. C. The FlavrSavr strain of tomato. D. A hybrid strain of corn produced from crossing two inbred strains of corn. The inbred strains were not transgenic. Answer: A. No. Dolly was not produced using recombinant techniques. There were not pieces of DNA that were cut and combined in a new way. B. Yes. C. Yes. D. No. The hybrids simply contain chromosomal genes from two different parental strains.

Q2. Describe the strategy for producing human proteins in the milk of livestock. Answer: Milk proteins are encoded by genes with promoters and regulatory sequences that direct the expression of these genes within the cells of the mammary gland. To get other proteins expressed in the mammary gland, the strategy is to link the promoter and regulatory sequences from a milk-specific gene to the coding sequence of the gene that encodes the human protein of interest. In some cases, it is also necessary to add a signal sequence to the amino terminal end of the target protein. A signal sequence is a short polypeptide that directs the secretion of a protein from a cell. If the target protein does not already have a signal sequence, it is possible to use a signal sequence from a milk-specific gene to promote the secretion of the target protein from the mammary cells and into the milk. During this process, the signal sequence is cleaved from the secreted protein.

Q3. Explain how one type of probe can be used to identify multiple bands in the Southern blot of a DNAfingerprinting experiment. Why do the lengths of these bands vary among different individuals? Answer: The reason why the same probe can identify multiple bands is because the probe is complementary to a genetic sequence that is located at several different sites throughout the genome. Not only is the sequence located at several different sites, it is also tandemly repeated at each site. Within populations, there is variation in the number of these tandem repeats at each site. If the number of tandem repeats at a given site is high, this will yield a higher molecular weight band (i.e., a longer band) in a Southern blot. This type of site is termed a VNTR (for variable number

Transgenic describes an organism that has had genes from another organism put into its genome for inheritance, through recombinant DNA techniques. A transgene is a gene or genetic material that has been transferred naturally or by any of a number of genetic engineering techniques from one organism to another. Transgenesis is the process of introducing an exogenous gene called a transgene into a living organism so that the organism will exhibit a new property and transmit that property to its offspring. Transgenesis can be facilitated by liposomes, plasmid vectors, viral vectors, pronuclear injection, protoplast fusion and ballistic DNA injection. Transgenic organisms are able to express foreign genes because the genetic code is similar for all organisms. This means that a specific DNA sequence will code for the same protein in all organisms.

Biological activity often not demonstrated in cells in culture

Several Post translational processings of cloned genes need an animal or plant host. Transgenics Proper processing and release in specific fluids like milk, urine or blood

Rich harvest / recovery & purification

Animals like cow, sheep, goat , pig etc. often are used for generation of transgenics May affect the health of generated transgenic animal Unsafe as virus or prions from animal or plant may affect human health

The method to generate transgenics and purification of the product is inefficient and expensive (~ $100,000/- for goat and ~ $500,000 /- for cow) Essential for production of certain proteins

Transgenic plant & plant cell culture Advantage : Edible antibody, vaccine etc. No need for rigorous purification of protein oral delivery of therapeutic protein Safe, as plant virus do not infect human Scale up easy - by increasing the acreage

Disadvantage: Low level expression (< 1% of total protein is considered as good) N- linked glycosylation is incomplete Certain mammalian post translational modifications missing Control of environment poor product vary from time to time, place to place Contamination of herbicides, insecticides, pesticides etc. High purity material is costly Current status: Few enzymes are produced from plants Production of Monoclonal antibodies Plant cell culture is being explored for safer, higher cell density, defined media & controllable

Edible Vaccines
Transgenic Plants Serving Human Health Needs
Works like any vaccine A transgenic plant with a pathogen protein gene is developed Potato, banana, and tomato are targets Humans eat the plant The body produces antibodies against pathogen protein Humans are immunized against the pathogen Examples: Diarrhea Hepatitis B Measles
NDSU

Why are transgenics important?

We can develop organisms that express a novel trait not normally found in the species

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

NDSU

Extension

Extension

Agriculture Transgenics in the Market

Insect resistant cotton Bt toxin kills the cotton boll worm transgene = Bt protein
Source: USDA

Some Ag Biotech Products Are Discontinued

Why??? Poor Quality FlavrSavr tomatoes (Calgene) Negative Consumer Response Tomato paste (Zeneca)

Insect resistant corn Bt toxin kills the European corn borer transgene = Bt protein
Normal Transgenic

Negative Corporate Response NewLeaf (Monsanto) Universal Negative Publicity StarLink corn (Aventis)
Extension

NDSU

NDSU

Extension

Next Generation of Ag Biotech Products

Golden Rice increased Vitamin A content (but not without controversy) transgene = three pathway enzymes

Sunflower white mold resistance transgene = oxalate oxidase from wheat

Source: Minnesota Microscopy Society

Turfgrass herbicide resistance; slower growing (= reduced mowing)

Bio Steel spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

Human Applications
Pharmaceutical products New solutions to old problems Disease diagnosis Determine what disease you have or may get Gene therapy Correcting disease by introducing a corrective gene

Biotechnology and Health

Product Insulin Interferon Interleukin Human growth hormone Neuroactive proteins
The genes for these proteins are:
Cloned Inserted into bacteria Product isolated using biofermentation

Use Diabetes Cancer Cancer Dwarfism Pain

Biotechnology and Health

Product Insulin Interferon Interleukin Human growth hormone Neuroactive proteins
The genes for these proteins are:
Cloned Inserted into bacteria Product isolated using biofermentation

Use Diabetes Cancer Cancer Dwarfism Pain

Environmental Applications
Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution

Indicator bacteria contamination can be detected in the environment

Future Health-related Biotech Products

Vaccines herpes, hepatitis C, AIDS, malaria

Tooth decay engineered Streptococcus mutans, the bacteria that destroys enamel

Transgenic production

Transgenic vs. knock-out

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 over expressing and mis-expressing endogenous or foreign genes at specific times and locations in the animals

Transgenic: an organism that has had DNA introduced into one or more of its cells artificially transgenic: DNA is integrated in a random fashion by injecting it into the pronucleus of a fertilized ovum
Random (approx.. 10% disrupt an endogenous gene important for normal development) multiple copies

Transgenic vs. knock-out

knockout: DNA is introduced first into embryonic stem (ES) cells. ES cells that have undergone homologous recombination are identified and injected into a 4 day old mouse embryo - a blastocyst
targeted insertion

METHODS OF TRANSGENESIS
Procedure:

Super-ovulation with FSH (source-Pregnant mares serum) for stimulating maturation of large number of ovarian follicles Surging the ovary with LH (source- Human Chorionic gonadotropin) for ovulation Mating with healthy male Select the mated females (checking the vaginal plug) Sacrifice the mated females and take out the fertilized eggs (up to 30 eggs/ female) Treatment of zygote with Hyaluronidase for removal of cumulus cells (Cumulus cells are specialized granulosa cells surrounding and nourishing the oocyte. These cells surround the fully-grown oocyte to form a cumulusoocyte complex (abbr. COC). Store zygotes in CO2 incubator at 37C Zygotes (25 to 30) are processed one by one Microinjection (needle dia 1 m) of foreign gene (DNA conc. 1-2 g/ml). Pierce the zona pellucida and the pronuclear nuclear membrane (highly elastic). Injected cells are kept in CO2 incubator at 37C o/n for dividing to 2 cell stage

Re-implant the 1 or 2 cell stage embryos in to anaesthetised & vasectomised (sterile) pseudopregnant females and allow the foetus to grow At weaning the Transgenic is tested for the presence of the foreign gene by PCR of blood or Southern Blot of tail biopsies.

Transgenic production
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

Brinster's growth hormone 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. (Dr. Anne Calof)

GFP transgenic mouse (Nagy)

9.5 day embryos GFP and wt

Tail tip

GFP transgenic mouse (Nagy)

Procedure: (Repeated) Super-ovulation with FSH (source-Pregnant mares serum) for stimulating maturation of large number of ovarian follicles Surging the ovary with LH (source- Human Chorionic gonadotropin) for ovulation Mating with healthy male Select the mated females (checking the vaginal plug) Sacrifice the mated females and take out the fertilized eggs (up to 30 eggs/ female)

Treatment of zygote with Hyaluronidase for removal of cumulus cells (Cumulus

cells are specialized granulosa cells surrounding and nourishing the oocyte. These cells surround the fully-grown oocyte to form a cumulus-oocyte complex (abbr. COC). Store zygotes in CO2 incubator at 37C Zygotes (25 to 30) are processed one by one Microinjection (needle dia 1 m) of foreign gene (DNA conc. 1-2 g/ml). Pierce the zona pellucida and the pronuclear nuclear membrane (highly elastic). Injected cells are kept in CO2 incubator at 37C o/n for dividing to 2 cell stage (to confirm viability) Re-implant the 1 or 2 cell stage embryos in to anaesthetised & vasectomised (sterile) pseudopregnant females and allow the foetus to grow At weaning the Transgenic is tested for the presence of the foreign gene by PCR of blood or Southern Blot of tail biopsies. If the foreign gene is incorporated at one cell stage (before cell division) then all the cells (germ line and somatic) will have the transgene. However, if the integration occurs after the 1st cell division than the product will be chimera of transgenics and non transgenics.

Planning a Transgenic production mouse colony

Mouse strain - popular Colony size
typical injection 200 embryos (7-10 females s.o.) Superovulation efficiency Parenting suitability Pseudo-pregs

Injecting fertilized eggs

The eggs are harvested 0.5 dpc (superovulated or natural matings) The DNA is usually injected into the male pronucleus The eggs can be transferred the same day or the next (2-cell) into pseudopregnant female oviducts

Pronuclear injection

Implantation of 1 or 2 cell embryos

The injected eggs are implanted the same day or are incubated overnight and implanted the next day Injected eggs are transferred to the oviduct of a 0.5 dpc (days post coitum) pseudopregnant female

Implanting 1(or 2) cell embryos

Implanting 1(or 2) cell embryos (cont.)

Pseudopregnant females and vasectomized males

Female mice can be tricked into thinking they are pregnant A mouse in estrus is mated with a vasectomized male pseudopregnancy If eggs (blastocysts) implanted will become truly pregnant and will give birth to live offspring

Vasectomizing

Breeding Tg founders
Individually backcrossed to the strain of choice DO NOT intercross different founders - each founder results from a separate RANDOM transgene integration even

Transgenic mice as tools

Study gene function
Many human diseases can be modeled by introducing the same mutation into the mouse. Intact organism provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing

Drug testing

Transgenic mice as tools

Polio virus receptor Normal mice can't be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. Tg mice expressing the human gene for the receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans

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. (knock out function!) Vector DNA to enable the molecules to be inserted into host DNA molecules

Typical KO vector

*tk:thymidine kinase

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 are left to rest for a couple of hours Expanded blastocysts are transferred to the uterine horn of a 2.5 dpc pseudopregnant female Max. 1/3 of transferred blasts will develop into healthy pups

Implanting blastocysts

Implanting blastocysts (cont.)

Littermates
Black mouse no apparent ES cell contribution

Chimeric founder strong ES cell contribution

Chimeric founder weaker ES cell contribution

Chimeric mouse

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 strain (agouti or white colour) and the ES cells are injected into a C57Bl/6 blastocyst (black). The more that the ES cells contribute to the genome of the mouse, the more the coat colour will be agouti. The chimera mouse is usually tiger striped.

Breeding Chimeras (knock-out founder)cont

Males that are 40% to 100% based on agouti coat colour should be bred Females should not be bred (low incidence of success) ES cells are male. Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting, not likely to go germline and should be sac'ed

Knock-out mice as tools

If the replacement gene is nonfunctional (null allele), mating of the heterozygous will produce a strain of "knock-outs' homozygous for the nonfunctional gene (both copies are knocked-out
Find out if the gene is indispensable (suprisingly many are not!) "pleiotropic" expression in different tissues in different ways and at different times in development

Breeding Transgenics
Most transgenics are bred onto a C57Bl/6 background
standard

BL/6 breeding information

mate 6-8 weeks for best reproductive performance replace males when 1 year old

Breeding Transgenics (cont.)

Replace females after 6 litters or at 6 months of age quick breeding - 1 founder male: 2 females rotation of females through male cage

Common problems:
female not good mother, check for milk - give auntie male cannibalizing litter fighting (separate) Do not reunite males

Breeding Transgenics (cont)

Stick to schedules or be overwhelmed
strict records (birth, ID, parents) ID pups tail tip or collect ear tissue at 2 weeks try to genotype before weaning wean only positives, sac negatives (mosaics?) house male and females separately mate at 6 weeks

Housing
Range from conventional to barrier Researcher can usually advise on level of protection that is appropriate

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!

Parasiticide - Ivermectin - immune systemmodulating activity

Pathogens (cont): 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 No carrier state - stop breeding Altered physiology: as the virus travels down the resp.. tract -necrosis of airway epithelium, pneumonia in lungs, lesions. 129/J and DBA, aged and immunodeficient 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 Isograft rejection Wound healing

Pathogens (cont): Some common pathogens and their effects

MHV
Probably most important pathogen of laboratory mice Extremely contagious; aerosol, direct contact; fomites 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 Immunocompromised 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, bilirubin concentration, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc. etc. etc.

Pathogens (cont.): Some common pathogens and their effects

Helicobacter spp
Genus keeps expanding with discoveries H. Hepaticus (mice) most prominent Transmission: direct fecal-oral or fomites Clinical signs absent in immunocompetent Immunodeficient - rectal prolapse Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms

Pathogens (cont.): Some common pathogens and their effects

Reported effects: confounds carcinogenicity research; gastointestinal system research

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

C57Bl very susceptible Infestation: asymptomatic or may cause wasting; scruffiness; pruritus; patchy alopecia; accumulation of fine bran-like material, mostly over affected areas; self-trauma to the point of amputation; and secondary pyoderma Pathological changes: hyperkeratosis, erythema, mast cell infiltration, ulcerative dermatitis, splenic lymphoid and lymph node hyperplasia;

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

Planning a Transgenic production mouse colony

Mouse strain - popular Colony size
typical injection 200 embryos (7-10 females s.o.) Superovulation efficiency Parenting suitability Pseudo-pregs

Injecting fertilized eggs

The eggs are harvested 0.5 dpc (superovulated or natural matings) The DNA is usually injected into the male pronucleus The eggs can be transferred the same day or the next (2-cell) into pseudopregnant female oviducts

Pronuclear injection

Implantation of 1 or 2 cell embryos

The injected eggs are implanted the same day or are incubated overnight and implanted the next day Injected eggs are transferred to the oviduct of a 0.5 dpc (days post coitum) pseudopregnant female

Implanting 1(or 2) cell embryos

Implanting 1(or 2) cell embryos (cont.)

Pseudopregnant females and vasectomized males

Female mice can be tricked into thinking they are pregnant A mouse in estrus is mated with a vasectomized male pseudopregnancy If eggs (blastocysts) implanted will become truly pregnant and will give birth to live offspring

Vasectomizing

Breeding Tg founders
Individually backcrossed to the strain of choice DO NOT intercross different founders - each founder results from a separate RANDOM transgene integration even

Transgenic mice as tools

Study gene function
Many human diseases can be modeled by introducing the same mutation into the mouse. Intact organism provides a more complete and physiologically relevant picture of a transgene's function than in vitro testing

Drug testing

Transgenic mice as tools

Polio virus receptor Normal mice can't be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. Tg mice expressing the human gene for the receptor can be infected by polio virus and even develop paralysis and other pathological changes characteristic of the disease in humans

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. (knock out function!) Vector DNA to enable the molecules to be inserted into host DNA molecules

Typical KO vector

*tk:thymidine kinase

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 are left to rest for a couple of hours Expanded blastocysts are transferred to the uterine horn of a 2.5 dpc pseudopregnant female Max. 1/3 of transferred blasts will develop into healthy pups

Implanting blastocysts

Implanting blastocysts (cont.)

Littermates
Black mouse no apparent ES cell contribution

Chimeric founder strong ES cell contribution

Chimeric founder weaker ES cell contribution

Chimeric mouse

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 strain (agouti or white colour) and the ES cells are injected into a C57Bl/6 blastocyst (black). The more that the ES cells contribute to the genome of the mouse, the more the coat colour will be agouti. The chimera mouse is usually tiger striped.

Breeding Chimeras (knock-out founder)cont

Males that are 40% to 100% based on agouti coat colour should be bred Females should not be bred (low incidence of success) ES cells are male. Breed aggressively- rotate females through male's cage. If the male produces more than 6 litters without transmitting, not likely to go germline and should be sac'ed

Knock-out mice as tools

If the replacement gene is nonfunctional (null allele), mating of the heterozygous will produce a strain of "knock-outs' homozygous for the nonfunctional gene (both copies are knocked-out
Find out if the gene is indispensable (suprisingly many are not!) "pleiotropic" expression in different tissues in different ways and at different times in development

Breeding Transgenics
Most transgenics are bred onto a C57Bl/6 background
standard

BL/6 breeding information

mate 6-8 weeks for best reproductive performance replace males when 1 year old

Breeding Transgenics (cont.)

Replace females after 6 litters or at 6 months of age quick breeding - 1 founder male: 2 females rotation of females through male cage

Common problems:
female not good mother, check for milk - give auntie male cannibalizing litter fighting (separate) Do not reunite males

Breeding Transgenics (cont)

Stick to schedules or be overwhelmed
strict records (birth, ID, parents) ID pups tail tip or collect ear tissue at 2 weeks try to genotype before weaning wean only positives, sac negatives (mosaics?) house male and females separately mate at 6 weeks

Housing
Range from conventional to barrier Researcher can usually advise on level of protection that is appropriate

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!

Parasiticide - Ivermectin - immune systemmodulating activity

Pathogens (cont): 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 No carrier state - stop breeding Altered physiology: as the virus travels down the resp.. tract -necrosis of airway epithelium, pneumonia in lungs, lesions. 129/J and DBA, aged and immunodeficient 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 Isograft rejection Wound healing

Pathogens (cont): Some common pathogens and their effects

MHV
Probably most important pathogen of laboratory mice Extremely contagious; aerosol, direct contact; fomites 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 Immunocompromised 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, bilirubin concentration, enhanced phagocytic activity of macrophages, rejection of xenograft tumors etc. etc. etc.

Pathogens (cont.): Some common pathogens and their effects

Helicobacter spp
Genus keeps expanding with discoveries H. Hepaticus (mice) most prominent Transmission: direct fecal-oral or fomites Clinical signs absent in immunocompetent Immunodeficient - rectal prolapse Pathological changes: chronic, active hepatitis, enterocolitis, hepatocellular neoplasms

Pathogens (cont.): Some common pathogens and their effects

Reported effects: confounds carcinogenicity research; gastointestinal system research

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

C57Bl very susceptible Infestation: asymptomatic or may cause wasting; scruffiness; pruritus; patchy alopecia; accumulation of fine bran-like material, mostly over affected areas; self-trauma to the point of amputation; and secondary pyoderma Pathological changes: hyperkeratosis, erythema, mast cell infiltration, ulcerative dermatitis, splenic lymphoid and lymph node hyperplasia;

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

Product Why??? Use Vaccines modifiedhepatitis C, AIDS, malaria herpes, organisms GMOs - any vaccine Works likeGenetically Insulin Diabetes first Bioremediation cleanup Transgenic Insect is introduced developed knockout: DNAresistant contaminated Transgenic: aan organism that has toxinDNA Poor Quality Disease are -often generated into Pharmaceutical mice Treatment cotton is Bthadtokills the A transgenicdevelop sites;pathogen protein genebiotechnology We can plant with products boll worm a novel trait organisms microbes designed to degrade that express of usesbasic Genetic engineering isold problemsCancer the introduced into Definition set FlavrSavr Interferon - tomatoes (ES) tool Biotechnologyorganism the(Calgene) traitsathat resultthat embryonic in cotton ability Potato, banana, andGeneralarespeciescells. ESpromoter to targets not New -solutions to that expresses of of its cells normally tomato the 1. characterizetransgene more cells GMO an foundstemone or = Bt protein the pollution Interleukin Humansartificially of Diagnosticsto improve eat the plant involves: gene expression The the introduction of homologous recombination application technology Cancer from have undergoneResponse Genetic engineering direct Consumer foreign Negativetissue-specific DNA Disease diagnosis The body a biological organism produces antibodies against Dwarfism pathogen Human growth hormone injected intoprotein are immunizedDNA is integrated a reporter identified against theattached a 4 a Tomato what disease Cleanup Determinepromoter can behave or mayorganism old transgenic: and you transgenic to indaymutans, e.g. apaste (Zeneca) engineered Streptococcus Environmentalto pathogen get Humans are Originally a Toothequivalent term NeuroactiveInsect Extended shelf-life Bt toxin can be Isolating such decay a blastocyst (Flavr-Savr) contamination proteins by injecting Pain mouse genes as bacteria corn Examples: geneIndicatorresistant destroys enamel kills the or GFP randomembryoLacZfunctiontomatoit into the fashion-Definition BiotechnologyCorporate they environment Detailed that Negative -detected inResponse better Gene therapyHuman Applications Modifyingthe bacteriathe borer genes so corn European Diarrhea NewLeaf (Monsanto) targeted insertion pronucleusthethe technology to improve 2. application of a introducing a expressing examine of be inserted into new Correctinggenes toeffects of overacorrective gene Preparing disease byfertilized ovumspecies and The = Bt Hepatitis B transgene are: protein The genes for these proteinsHerbicide resistant soybean (Roundup Ready) mis-expressing endogenous or Developing transgenes 10% organisman endogenous the biological function of an disruptforeign genes at Random (approx.. Measles Universal Negative and locations in the animals Normal Cloned Transgenic specific times Publicity by adding genes from another organisms gene important Inserted into bacteria for normal development) StarLink corn (Aventis) Product isolated using biofermentation multiple copies
Source: USDA

Future Biotechnology theinHealth Health-related Needthe Product Biotech Transgenics are a BiotechnologyMarket A Popular EdibleWe andTerm Know What Products Term Vaccines Agriculture Transgenics AreToProducts Environmental ApplicationsFarm Some Ag Biotech Aboutknock-out Why are transgenics important?Needs Humanvs. Human Health Applicationsthe Biotechnology Serving Just onDiscontinued isproduction Not Transgenic Transgenic Transgenic Genetic Engineering? Plants How about some definitions

Transgenic describes an organism that has had genes from another organism put into its genome for inheritance, through recombinant DNA techniques.
GloFish, the first genetically modified animal to be sold as a pet

A genetically modified organism (GMO) or genetically engineered organism (GEO) is an organism whose genetic material has been altered using genetic engineering techniques. These techniques, generally known as recombinant DNA technology, use DNA molecules from different sources, which are combined into one molecule to create a new set of genes. This DNA is then transferred into an organism, giving it modified or novel genes. Transgenic organisms, a subset of GMOs, are organisms that have inserted DNA from a different species. GMOs are the constituents of genetically modified foods.

NDSU NDSU NDSU NDSU NDSU NDSU NDSU NDSU NDSU NDSU NDSU NDSU

Extension

The procedure: Hormones used:Gonadotropins: Luteinizing Hormone (LH) and Follicle Stimulating Hormones (FSH)

Luteinizing hormone (LH) [from HCG] and follicle-stimulating hormone (FSH) [from PMS] are called the gonadotropins because they stimulate the gonads (in males, the testes, and in females, the ovaries). They are not necessary for life, but are essential for reproduction. These two hormones are secreted from cells in the anterior pituitary called gonadotrophs. Most gonadotrophs secrete only LH or FSH, but some appear to secrete both hormones. Like thyroid-simulating hormone, LH and FSH are large glycoproteins composed of alpha and beta subunits. The alpha subunits are identical in all three of these anterior pituitary hormones, while the beta subunits are unique and endows each hormone with the ability to bind its own receptor. Physiologic Effects of Gonadotropins Physiologic effects of the gonadotrophins are known only in the ovaries and testes. Together, then regulate many aspects of gonadal function in both males and females. Luteinizing Hormone In both sexes, LH stimulates secretion of sex steroids from the gonads. In the testes, LH binds to receptors on Leydig cells, stimulating synthesis and secretion of testosterone. In the ovary, the Theca cells respond to LH stimulation by secretion of testosterone, which is converted into estrogen by adjacent granulosa cells. In females, ovulation of mature follicles on the

ovary is induced by a large burst of LH secretion known as the preovulatory LH surge. Residual cells within ovulated follicles proliferate to form corpora lutea, which secrete the steroid hormones progesterone and estradiol. Progesterone is necessary for maintenance of pregnancy, and, in most mammals, LH is required for continued development and function of corpora lutea. The name luteinizing hormone derives from this effect of inducing luteinization of ovarian follicles. LH is obtained from Human chorionic gonadotropin

Human chorionic gonadotropin or human chorionic gonadotrophin (hCG) is a glycoprotein hormone produced in pregnancy that is made by the developing embryo after conception and later by the syncytiotrophoblast (part of the placenta), but it is not known whether this production is a contributing cause or an effect of tumorigenesis.

Human chorionic gonadotropin or human chorionic gonadotrophin (hCG) is a glycoprotein hormone produced in pregnancy that is made by the developing embryo after conception and later by the syncytiotrophoblast (part of the placenta), but it is not known whether this production is a contributing cause or an effect of tumorigenesis.

Structure Human chorionic gonadotropin is a glycoprotein composed of 244 amino acids with a molecular mass of 36.7 kDa. Its total dimensions are 753530 ngstrms (7.53.53 nanometers). It is heterodimeric, with an (alpha) subunit identical to that of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and (beta) subunit that is unique to hCG. The (alpha) subunit is 92 amino acids long and has dimensions 602515 ngstrms (62.51.5 nm). The -subunit of hCG gonadotropin contains 145 amino acids and has dimensions 6.52.52 nm, encoded by six highly-homologous genes that are arranged in tandem and inverted pairs on chromosome 19q13.3 - CGB (1, 2, 3, 5, 7, 8). The two subunits create a small hydrophobic core surrounded by a high surface area-to-volume ratio: 2.8 times that of a sphere. The vast majority of the outer amino acids are hydrophilic.

Function: Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly-negative charge, hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. It has also been hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. For example, hCG-treated endometrial cells induce an increase in T cell apoptosis (dissolution of T-cells). These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance, and may facilitate the trophoblast invasion, which is known to expedite fetal development in the endometrium. It has also been suggested that hCG levels are linked to the severity of morning sickness in pregnant women. Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes. As the most abundant biological source is women who are presently pregnant, some organizations collect urine from pregnant women to extract hCG for use in fertility treatment. Human chorionic gonadotropin also plays a role in cellular differentiation/proliferation and may activate apoptosis.

Follicle-Stimulating Hormone As the name implies, FSH stimulates the maturation of ovarian follicles. Administration of FSH to human and animals induces "superovulation", or development of more than the usual number of mature follicles and hence, an increased number of mature gametes.
FSH is also critical for sperm production. It supports the function of Sertoli cells, which in turn support many aspects of sperm cell maturation. Control of Gonadotropin Secretion The principal regulator of LH and FSH secretion is gonadotropin-releasing hormone (GnRH), also known as LH-releasing hormone (LHRH). GnRH is a ten amino acid peptide that is synthesized and secreted from hypothalamic neurons and binds to receptors on gonadotrophs. As depicted in the figure to the right, GnRH stimultes secretion of LH, which in turn stimulates gonadal secretion of the sex steroids testosterone, estrogen and progesterone. In a classical negative feedback loop, sex steroids inhibit secretion of GnRH and also appear to have direct negative effects on gonadotrophs.

This regulatory loop leads to pulsatile secretion of LH and, to a much lesser extent, FSH. The number of pulses of GnRH and LH varies from a few per day to one or more per hour. In females, pulse frequency is clearly related to stage of the cycle. Luteinizing hormone (LH) [from HCG] and follicle-stimulating hormone (FSH) [from PMS] are called the gonadotropins because they stimulate the gonads (in males, the testes, and in females, the ovaries). They are not necessary for life, but are essential for reproduction. These two hormones are secreted from cells in the anterior pituitary called gonadotrophs. Most gonadotrophs secrete only LH or FSH, but some appear to secrete both hormones. Like thyroid-simulating hormone, LH and FSH are large glycoproteins composed of alpha and beta subunits. The alpha subunits are identical in all three of these anterior pituitary hormones, while the beta subunits are unique and endows each hormone with the ability to bind its own receptor. Physiologic Effects of Gonadotropins Physiologic effects of the gonadotrophins are known only in the ovaries and testes. Together, then regulate many aspects of gonadal function in both males and females. Luteinizing Hormone In both sexes, LH stimulates secretion of sex steroids from the gonads. In the testes, LH binds to receptors on Leydig cells, stimulating synthesis and secretion of testosterone. In the ovary, the Theca cells respond to LH stimulation by secretion of testosterone, which is converted into estrogen by adjacent granulosa cells. In females, ovulation of mature follicles on the ovary is induced by a large burst of LH secretion known as the preovulatory LH surge. Residual cells within ovulated follicles proliferate to form

Luteinizing hormone (LH) [from HCG] and follicle-stimulating hormone (FSH) [from PMS] are called the gonadotropins because they stimulate the gonads (in males, the testes, and in females, the ovaries). They are not necessary for life, but are essential for reproduction. These two hormones are secreted from cells in the anterior pituitary called gonadotrophs. Most gonadotrophs secrete only LH or FSH, but some appear to secrete both hormones. Like thyroid-simulating hormone, LH and FSH are large glycoproteins composed of alpha and beta subunits. The alpha subunits are identical in all three of these anterior pituitary hormones, while the beta subunits are unique and endows each hormone with the ability to bind its own receptor. Physiologic Effects of Gonadotropins Physiologic effects of the gonadotrophins are known only in the ovaries and testes. Together, then regulate many aspects of gonadal function in both males and females. Luteinizing Hormone In both sexes, LH stimulates secretion of sex steroids from the gonads. In the testes, LH binds to receptors on Leydig cells, stimulating synthesis and secretion of testosterone. In the ovary, the Theca cells respond to LH stimulation by secretion of testosterone, which is converted into estrogen by adjacent granulosa cells. In females, ovulation of mature follicles on the ovary is induced by a large burst of LH secretion known as the preovulatory LH surge. Residual cells within ovulated follicles proliferate to form corpora lutea, which secrete the steroid hormones progesterone and estradiol. Progesterone is necessary for maintenance of pregnancy, and, in most mammals, LH is required for continued development and function of corpora lutea. The name luteinizing hormone derives from this effect of inducing luteinization of ovarian follicles. LH is obtained from Human chorionic gonadotropin

Follicle-Stimulating Hormone As the name implies, FSH stimulates the maturation of ovarian follicles. Administration of FSH to human and animals induces "superovulation", or development of more than the usual number of mature follicles and hence, an increased number of mature gametes. FSH is also critical for sperm production. It supports the function of Sertoli cells, which in turn support many aspects of sperm cell maturation. Control of Gonadotropin Secretion The principal regulator of LH and FSH secretion is gonadotropin-releasing hormone (GnRH), also known as LH-releasing hormone (LHRH). GnRH is a ten amino acid peptide that is synthesized and secreted from hypothalamic neurons and binds to receptors on gonadotrophs. As depicted in the figure to the right, GnRH stimultes secretion of LH, which in turn stimulates gonadal secretion of the sex steroids testosterone, estrogen and progesterone. In a classical negative feedback loop, sex steroids inhibit secretion of GnRH and also appear to have direct negative effects on gonadotrophs.

This regulatory loop leads to pulsatile secretion of LH and, to a much lesser extent, FSH. The number of pulses of GnRH and LH varies from a few per day to one or more per hour. In females, pulse frequency is clearly related to stage of the cycle. Numerous hormones influence GnRH secretion, and positive and negative control over GnRH and gonadotropin secretion is actually considerably more complex than depicted in the figure. For example, the gonads secrete at least two additional hormones - inhibin and activin - which selectively inhibit and activate FSH secretion from the pituitary. Pregnant mare serum gonadotropin (PMSG) Originates in the endometrial cups and present in the blood during the period 40 to 140 days of pregnancy. Used pharmaceutically to stimulate growth of follicles in inactive ovaries in adult animals and in combination with prostaglandin to induce superovulation in cows which are acting as donors for embryo transfers. Now called equine chorionic gonadotropin (eCG). Hyaluronidase Pronunciation: (HYE-al-ure-ON-i-dase) Class: Physical adjunct Trade Names: Amphadase - Solution for injection 150 units/mL (bovine source) Trade Names: Hylenex - Solution for injection 150 units/mL (recombinant human)

Trade Names: Vitrase - Solution for injection 200 units/mL (ovine source) - Powder for injection, lyophilized 6,200 units (ovine source)

Human chorionic gonadotropin or human chorionic gonadotrophin (hCG) is a glycoprotein hormone produced in pregnancy that is made by the developing embryo after conception and later by the syncytiotrophoblast (part of the placenta), but it is not known whether this production is a contributing cause or an effect of tumorigenesis. Structure Human chorionic gonadotropin is a glycoprotein composed of 244 amino acids with a molecular mass of 36.7 kDa. Its total dimensions are 753530 ngstrms (7.53.53 nanometers). It is heterodimeric, with an (alpha) subunit identical to that of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and (beta) subunit that is unique to hCG. The (alpha) subunit is 92 amino acids long and has dimensions 602515 ngstrms (62.51.5 nm). The -subunit of hCG gonadotropin contains 145 amino acids and has dimensions 6.52.52 nm, encoded by six highly-homologous genes that are arranged in tandem and inverted pairs on chromosome 19q13.3 - CGB (1, 2, 3, 5, 7, 8). The two subunits create a small hydrophobic core surrounded by a high surface area-to-volume ratio: 2.8 times that of a sphere. The vast majority of the outer amino acids are hydrophilic.

Function Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly-negative charge, hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. It has also been hypothesized that hCG may be a placental link for the development of local maternal immunotolerance. For example, hCG-treated endometrial cells induce an increase in T cell apoptosis (dissolution of T-cells). These results suggest that hCG may be a link in the development of peritrophoblastic immune tolerance, and may facilitate the trophoblast invasion, which is known to expedite fetal development in the endometrium. It has also been suggested that hCG levels are linked to the severity of morning sickness in pregnant women. Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes. As the most abundant biological source is women who are presently pregnant, some organizations collect urine from pregnant women to extract hCG for use in fertility treatment. Human chorionic gonadotropin also plays a role in cellular differentiation/proliferation and may activate apoptosis.