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Creating Transgenic Animals

Transgenic - introducing a gene from one species into another species

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- the newly fertilized egg still has a sperm pronucleus and an egg pronucleus - foreign DNA can randomly or specifically integrate into the genome - It will be present on one chromosome in all the cells of the offspring

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Creating transgenic mice

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Creating transgenic mice

- can knockout genes (delete them) and study the phenotype caused

- can modify gene in some way and put them back in and study the effect

- can alter genes to mimic human diseases and build a mouse model

- mice are the major model system for studying human development and disease 50

Creating transgenic mice

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Producing Proteins or Drugs in Transgenic Animals

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expressed in mammary tissue

Producing Proteins or Drugs in Transgenic Animals

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2009 - antithrombin protein (anti blood clotting drug) approved by this method

- produced in a herd of goats in Massachusetts

- one goat can produce as much drug in its milk in a year as would be purified from 90,000 blood donations

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Genetically-modified fish

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Wild type salmon

AquaBounty salmon

- 18 years and still waiting in the US

- production was recently approved in Canada

- still need approval for consumption of fish

- a large number of US stores have already pledged not to sell it

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- one method to make transgenic plants uses the Ti plasmid to introduce foreign DNA

- Ti plasmid is from a bacteria called Agrobacterium tumefaciens that naturally infects plant cells and transfers DNA into them!

Making transgenic plants

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Making transgenic plants

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Making transgenic plants

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About 12% of the worlds usable land is currently planted with transgenic plants

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Producing Bt-expressing plants: pest resistance

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- first used in 1996

- now more than 90% of our soybeans and 60% of our corn has been modified to express the Bt gene

Producing Bt-expressing plants: pest resistance

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Creating Roundup Ready plants

- farmers spend a lot of money on weedkiller to kill weeds in their crop fields

- the active ingredient in the weedkiller Roundup is glyphosate

- glycophosate inhibits an enzyme (ESPS) in the plant chloroplast required to make several amino acids

- unfortunately, both crop plants and weeds are sensitive to the weedkiller

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- however, there is a bacterial ESPS enzyme that is resistant to the effects of glyphosate

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Creating Roundup Ready plants

- introduce into crop plants a genetically-engineered form of the bacterial ESPS enzyme that is resistant to glyphosate

- the crop plants are now Roundup ready

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Dead weeds cant become resistant some Monsanto scientist

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Golden Rice - introduced six foreign genes!

- rice is a major plant staple in large parts of the world, but it has a few problems nutritionally

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Golden Rice - introduced six foreign genes!

1) beta-carotene is made by green vegetables and is converted into vitamin A

- vitamin A deficiency results in increased disease susceptibility and even blindness, and increases childhood mortality

- rice cannot synthesize beta-carotene

- therefore vitamin A deficiency is widespread where rice is consumed

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Golden Rice - introduced six foreign genes!

2) iron is needed to make heme, a major component of hemoglobin, andhumans cannot make heme but must get it from their diet

- rice makes phytate, a compound that binds iron and interferes with its absorption in the intestine

- therefore, iron deficiency (anemia) is another major problem in areas of high rice consumption

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Golden Rice - introduced six foreign genes!

1) rice cannot synthesize beta-carotene

- engineered into rice the genes for four enzymes needed to make beta-carotene (two from daffodil, two from bacteria)

2) rice makes phytate, which binds iron

- engineered into rice genes to make a bacterial enzyme that breaks down phytate, and a bean protein that helps with iron absorption

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Golden Rice - introduced six foreign genes!

- bad news

- you have to eat all of your daily rice as golden rice to get the recommended amount of beta-carotene

- after 14 years, it is still not being grown anywhere (it is being tested in the Phillipines)

- there is significant opposition to it by people and governments in Asia and Africa

- http://www.npr.org/blogs/thesalt/2013/03/07/173611461/in-a-grain-of-golden-rice-a-world-of-controversy-over-gmo-foods

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general structure of a gene

- the upstream region in DNA contains information which indicates where to start transcribing - also when to transcribe the gene (under what conditions)

this region is transcribed

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Regulation of eukaryotic genes is more complex

- a typical eukaryotic gene is regulated by multiple transcription factors

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Methods to study where and when a gene or protein is expressed

1) perform antibody staining - make an antibody to yourprotein in rabbits or mice, purify that antibody and use it tofind where and when the protein is made using fixed cellsor tissues

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- expensive, takes time, cells are fixed

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Methods to study where and when a gene or protein is expressed

2) perform in situ hybridization make a labeled probecomplementary to the mRNA and use it to find where andwhen the mRNA is produced in fixed cells or tissues

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- resolution is so-so, cells are fixed

Methods to study where and when a gene or protein is expressed

3) make a reporter construct fuse the upstream regionof your gene of interest to the coding sequences for green fluorescent protein and look where and when GFP is expressed in living animals

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Many marine organisms show bioluminescence

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The Nobel Prize in Chemistry 2008 Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

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jellyfish green fluorescent protein = GFP

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When blue light shines on this protein, it emits green light back

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The Nobel Prize in Chemistry 2008 Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

Can GFP be used in other organisms to make reporter constructs?

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your favorite gene promoter for YFG

Reporter constructs made with GFP can show where and when a gene or protein is expressed

promoter for YFG

promoter for YFG YFG ORF

A transcriptional reporter makes GFP whenever and wherever the normal gene is expressed

A translational reporter makes GFP whenever and wherever the normal protein is expressed (protein fusion)

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The Nobel Prize in Chemistry 2008 Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

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The Nobel Prize in Chemistry 2008 Roger Y. Tsien, Martin Chalfie, Osamu Shimomura

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Reporter constructs made with GFP can show where and when a gene or protein is expressed

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Retroviral Vectors Have Been Used in Gene Therapy

- molecular biology techniques are used to introduce a human gene into a retrovirus that infects human cells, and use the recombinant virus to carry the gene into the patients cells 88

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1) the wild type copy of the gene defective in the disease isinserted into a retroviral vector RNA genome

2) the recombinant viruses are used to infect defective cellstaken from the patient

3) the viral RNA is converted to DNA by reverse transcriptase

4) integration of the viral DNA introduces the wild type geneinto the genome of the patients cells

5) these cells are grown in tissue culture and reintroduced intothe patients body

Retroviral vector-based gene therapy

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- retroviral-based somatic cell gene therapy has been used to treat severe combined immune deficiency (SCID)

- mutations in several different genes can result in loss of both arms of the immune system - B cells and T cells - leaves patients completely sensitive to all infections (bubble boy)

- ADA SCID (adenine deaminase) - X-linked SCID (gamma chain of IL receptor)

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Retroviral vector-based gene therapy: successes

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Retroviral vector-based gene therapy: successes

- mutations in several different genes can result in loss of both arms of the immune system - B cells and T cells - leaves patients completely sensitive to all infections (bubble boy)

- ADA SCID (adenine deaminase) - X-linked SCID (gamma chain of IL receptor)

1990 - patient at NIH is given the first gene therapy - viruses with a good copy of the adenine deaminase gene are used to infect her T cells - she is still alive and gets a weekly shot of the virus

2000 - X-linked SCID patients are cured when their immune systems are reconstituted with recombinant viruses carrying the good copy of the gene

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2000 - X-linked SCID patients are cured when their immune systems are reconstituted with recombinant viruses carrying the good copy of the gene

- 5 of 20 patients in France and England develop leukemia - integration of virus upstream of an oncogene? - extra copies of the good gene cause cancer?

- trials of this method were halted in 2003 in the US

Retroviral vector-based gene therapy: problems

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Other viral vectors can be used in gene therapy

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Adenoviral vectors can be used in gene therapy

1) the wild type copy of the gene defective in the disease isinserted into an adenovirus (cold virus) genome

2) the recombinant viruses are exposed to defective cells takenfrom the patient

3) the viral genome does not integrate into the host genome

4) these cells can be grown in tissue culture and reintroducedinto the patients body

5) the wild type gene is transcribed and translated

- pro - lack of viral integration reduces risk of insertional mutagenesis - con - lack of viral integration means constant injections of cells

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Adenoviral vectors can be used in gene therapy

- 1999, Philadelphia

- 18 year old Jessie Gelsinger had a rare form of liver disease due to mutation of a gene on the X chromosome

- he received an injection of adenovirus containing the wild type version of the gene and died 4 days later due to a massive immune response to the virus

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Adenoviral vectors can be used in gene therapy

- 2007 British researchers use adenoviral gene therapy to treat an inherited retinal disease caused by mutations in a single gene

- 28/30 patients had an increase in vision and no apparent side-effects

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Newer methods being tested for gene therapy

Other methods to introduce wild type genes continue to be tested in animal models

- lipid bilayers - electroporation - gene gun bombardment

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Newer methods being tested for gene therapy

- US researchers used viral gene therapy to introduce a red opsin gene into eyes of adult dichromat squirrel monkey males

- treated males displayed trichromatic vision as shown by behavior tests

- precursor to a treatment for color blindness in humans

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