PRODUCTION OF TRANSGENIC ORGANISM

NETHRAVATHI RGN113011

III sem, MSc.Genetics

Transgenics technique permits the introduction of foreign genes or altered forms of an endogenous gene into an organism.

Mostly, this technique does not result in replacement of the endogenous gene, but rather the integration of additional copies of it.

The introduced gene is called TRANSGENE.

The organism carrying it is referred to as TRANSGENIC ORGANISM. (A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome.)

• TRANSGENESIS is the process by which yeilds stable introduction of a gene into another organism.

• The procedure for introducing exogenous donor DNA into a recipient cell is called TRANSFECTION.

• Foreign genes are inserted into the germ line of the animal, so it can be transmitted to the progeny.

INTRODUCTION TO TRANSGENICS

Unicellular organisms (such as bacteria or yeast)

(all cells transformed -> transgenic)

Multicellular organisms (such as animals, plants,..)

difference between:

manipulation of single cells -> cell line

(eg: expression in insect cells or mammalian cells)

manipulation of a whole plant or animal -> transgenic

(eg; can have a transgenic offspring!!!)

more difficult and expensive to create whole modified organism (transgenic) than just cell line!!!

INTRODUCTION TO TRANSGENICS…

IMPORTANCE OF TRANSGENIC ANIMALS

MEDICAL IMPORTANCEDisease modelBioreactors for pharmaceuticalsXenotransplantation

AGRICULTURAL IMPORTANCEDisease resistant animalsFor improving quality and quantity of milk, meat, eggs and wool production.

INDUSTRIAL IMPORTANCEToxicity sensitive transgenic animals to test chemicals.Spider silk in milk of goat

FIRST TRANSGENIC ANIMAL

a) by Ralph Brinster (U Pennsylvania) and Richard Palmiter (University of Washington) in 1982.

b) It was created by inserting a human growth hormone gene in mouse genome.

c) The offspring was much larger than the parents.

SUPERMOUSE

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 misexpressingendogenous or foreign genes at specific times and locations

PRODUCTION OF TRANSGENIC ORGANISM

STEP 1 – CONSTRUCTION OF A TRANSGENE

Transgene made of 3 parts:

• Promoter

• Gene to be expressed

• Termination sequence

STEP 2 – INTRODUCTION OF FOREIGN GENE INTO THE ANIMAL

– Pronuclear microinjection method

– Embryonic stem cell method.

PRODUCTION OF TRANSGENIC ANIMALS – THE METHADOLOGY

• Transgenic animals can be created by manipulating embryonic stem cells.

• ES cells are obtained from the inner cell mass of a blastocyst.

• Transgene is incorporated into the ES cellby

1. Microinjection2. By a retro virus3. By electroporation

• Transgenic stem cells are grown in vitro.

• Then they are inserted into a blastocyst and implanted into a host’s uterus to grow normally.

EMBRYONIC STEM CELL METHOD

• A female animal is superovulated and eggs are collected.

• The eggs are fertilized in vitro.

• The transgene containing solution

is injected into the male pronucleus

using a micropipette.

• Eggs with the transgenes are kept overnight in an incubator to develop to a 2 cell stage.

• The eggs are then implanted into the uterus of a pseudo -pregnant female (female which has been mated with a vasectimized male the previous night)

MICROINJECTION METHOD

BLASTOCYST MICROINJECTION

ELECTROPORATION

• Cells are subjected to a brief electric shock of several thousand volts

• become transiently permeable to DNA.

• Presumably the shock briefly opens holes in the cell membrane allowing the DNA to enter the cells before the holes reseal

DNA INCORPORATION in the cell

Once the foreign DNA is inside the host cell, enzymes that function normally in DNA repair and recombination join the fragments of foreign DNA into the host cells chromosome

The new fragment can either replace an endogenous gene-homologous recombination or it can remain as an independent extrachromosomal DNA molecule referred to as an episome.

IDENTIFICATION OF TRANSGENIC CELLS

• Since only a relatively small fraction of cells take up DNA, a selective technique must be available to identify the transgenic cells

• In most cases the exogenous DNA includes two additional genes

• The small fraction of cells in which homologous recombination takes place can be identified by a combination of positive and negative selection

POSITIVE AND NEGATIVE SELECTION

Positive Selection- One of the additional genes (neoR) confers neomycin resistance; it permits positive selection of cells in which either homologous (specific) or non-homologous (random) recombination has occurred.

Negative selection- The second gene, thymidine kinase gene from Herpes Simplex Virus (tkHSV) confers sensitivity to gancyclovir (a cytotoxic nucleotide analog). This gene permits negative selection of ES cells in which non-homologous recombination has occurred.

Only ES cells that undergo homologous recombination (i.e. gene-targeted specific insertion of the DNA construct) can survive this selection scheme.

Positive and Negative selection of recombinant ES cells

Recombinats with random insertion Nonrecombinat cell

Recombinats with gene-targeted insertion

Treat with neomycin(positive selection)

Treat with gancyclovir(negative selection)

APPLICATION OF TRANSGENIC ORGANISM

Transgenic animals as drug factories

• Desirable to have the protein drug secreted in an easily retrievable manner.

• A recombinant drug, secreted into the milk of the transgenic animal could be produced in large quantities and easily retrieved from the animal.

• Attach the promoter sequence of a major milk protein upstream of the drug gene.

• Although this foreign gene will be present in all of the cells of the transgenic animal it will only be expressed in the mammary tissue.

How may mammary tissue be used to produce recombinant protein?

Human Protein C

• Blood protein.

• Functions to control blood clotting.

• Some individuals have inborn deficiency require exogenous Protein C.

“Genie” The first genetically engineered animal to produce a human protein drug (human protein C) in her milk.

Genie

• Produced sufficient quantities of Human Protein C.

• 1 g of Human Protein C per 1 liter of milk.

• 200-times more than present in human blood.

1. Growth hormone (gigantism / dwarfism) -- goat

2. Human fertility hormones – cow

3. Fibrinogen – for burn patients -- sheep

Some examples of therapeutic protein production using transgenic animals

ANDi, the first transgenic primate born in January, 2000

“ANDi” stands for “inserted DNA” spelled backwards.

224 unfertilized rhesus eggs were infected with a GFP virus

~Half of the fertilized eggs grew and divided

40 were implanted into twenty surrogate mothers

five males were born, two were stillborn.

ANDi was the only live monkey carrying the GFP genehttp://www.ohsu.edu/unparchive/2001/011001andi.shtml

TRANSGENIC PRIMATE

TOBACCO PLANT

“Enviropigs”• Transgenic pigs expressing the phytase

gene in their salivary glands.

• The phytase gene was introduced via DNA microinjection and used the parotid secretory protein promoter to specifically drive expression in the salivary glands.

• Phytate is the predominant storage form of phosphorus in plant-based animal feeds (e.g., soybean meal)

• Pigs and poultry cannot digest phytateand consequently excrete large amounts of phosphorus

• “Enviro-pigs” excrete 75% less phosphorus.

• Microinjected an E. coli phytase gene under the control of a mouse parotid secretory protein promoter.

EnviropigTM an environmentally friendly breed of pigs that utilizes plant phosphorus efficiently.

In 1994, the first genetically engineered food item went the market when the FDA approved the sale of a tomato that had been genetically modified to stay riper longer off the vine.

TRANSGENIC TOMATO

Genetically modified strain of malaria-resistant

The creation of mosquitoes with green fluorescent testicles will help curb the spread of malaria carrying mosquitoes.

• South Korean scientists have successfully cloned cats

• to enable them to glow in the dark

• cats appear normal in visible light

• but their skins glow red under ultraviolet light

TRANSGENIC CATS

TRANSGENIC MICE

GENE CODING FOR A GROWTH HORMONE

TRANSGENIC MICE

CHIMERIC MOUSE

Black mouse -no apparent ES cell contribution

Chimeric founder -strong ES cellcontribution

Chimeric founder -weaker ES cellcontribution

• 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)

Tail tip

9.5 day embryos - GFP and wt

GFP transgenic mouse (Nagy)

TRANSGENIC MOUSE

ALZHEIMER’S MOUSE In the brain of Alzheimer’s

patients, dead nerve cells are entangled in a protein called amyloid.

Mouse made by introducing amyloid precursor gene into fertilized egg of mice.

ONCOMOUSE• Mouse model to study

cancer• Made by inserting

activated oncogenes.

SMART MOUSE Biological model engineered to

overexpress NR2B receptor in the synaptic pathway.

This makes the mice learn faster like juveniles throughout their lives.

TRANSGENIC ARTWORK (2000)

ALBA, The EGFP (enhanced GFP) bunny

http://www.ekac.org/gfpbunny.html#gfpbunnyanchor

http://news.aol.com/story/_a/glowing-pig-passes-genes-to-piglets/20080109143909990001?ncid=NWS00010000000001

TRANSGENIC PIGS

GloFish, originally developed in Singapore as a way to monitor water pollution

Produce by integrating a fluorescent protein gene from jelly fish into embryo of fish.

The normally black+-and-silver zebrafish was turned green or red by inserting various versions of the GFP gene

Glofish are on sale throughout the US except in California

Glofish retail for about $5 per fish. Normal zebrafish cost around one tenth of the price

http://www.nus.edu.sg/corporate/research/gallery/research12.htm

TRANSGENIC - GloFish

TRANSGENIC FISHES

SUPERFISH

Increased growth and size

Growth hormone gene inserted into fertilized egg.

Transgenic salmon grows about 10 – 11 times faster than normal fish.

And now there is pet cloning for a “small” fee…

Nine-week-old "Little Nicky" peers out from her carrying case in Texas. Little Nicky, a cloned cat, was sold to its new owner by Genetic Savings and Clone for $50,000 in December 2004.

August 07, 2008 | Bernann McKinney with one of the 5 puppies cloned from Booger, her late pet pit bull. It cost her $50,000. When Booger was diagnosed with cancer, a grief-stricken McKinney sought to have him cloned -- first by the now-defunct Genetic Savings and Clone, and then by South Korean company RNL Bio.

PIG FOR ORGAN TRANSPLANT

Pigs with human genes, in order to decrease the chance of organ rejection by human body.

TRANSGENIC GOATS ENGINEERED TO PRODUCE HUMAN BREAST MILK ( journal transgenic research, august 2012)

University of California scientists created the transgenic goats by transferring human genes for breast milk enzymes and proteins into goat embryos.

Produce 60 percent of the lysozyme and lactoferrin found in human mother’s milk.

For babies of mothers who aren’t present, or can’t nurse them, milk from these transgenic goats could provide the next-best alternative.

BIOLUMINESCENT TRANSGENIC MOUSE (PLoS ONE, aug 2012)

Research done by Cancer Biology and Therapeutics Group, Ireland.

Model for study mammary gland tumour development.

• Blurring the lines between species by creating transgenic combinations.

• There may be health risks associated with transgenics.

• There may be long term effects on the environment when transgenic animals are released into the field.

• Various bioethicist argue that it is wrong to create animals that would suffer as a result of genetic alteration.

ISSUES RELATED TO TRANSGENIC TECHNOLOGY

• Transgenic technology is a field that is under constant evolution.

• Many transgenic animals have been successfully created for a variety of purposes, and the prospects are enormous.

• It holds great potential in many fields including agriculture, medicine and industry.

• With proper research and careful use the transgenic animals can go a long way in solving several problems for which science doesn’t have a solution till now.

CONCLUSION

• Satyanarayana U, Biotechnology (2010), 1st edition, Books and allied (P) Ltd, Kolkata.

• Channarayappa, Molecular Biotechnology – Principles and Practices (2006), 1st edition, University Press Pvt Ltd, Hyderabad.

• Smith E John, Biotechnology (1996), 3rd edition, Cambridge University Press

• http://dsc.discovery.com/technology/tech-10/genetic-engineering/10-transgenic-animals-03.html

• www.biomedcentral.com/1471-2407/12/209

• http://news.e-healthsource.com/index.php?p=news1&id=534197

BIBLIOGRAPHY

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