chapter 16 molecular biology is expanding its reach

Chapter 16Chapter 16Molecular Biology is Molecular Biology is Expanding Its ReachExpanding Its Reach

Kuang-Hui Lu:Kuang-Hui Lu:

Uses of Recombinant DNA Uses of Recombinant DNA Technology in ResearchTechnology in Research

• Creating bacteria and other organisms capable of synthesizing both useful and economically important molecules.

• Mapping the genomes of humans and of organisms utilized in research.

• Supplying DNA and RNA sequences as research tools.

• Altering the genotype of organisms (both plants and animals) (transgenesis)

• Potentially correcting genetic defects in animals (gene therapy)

Mammals Made to OrderMammals Made to Order

• It is difficult to isolate an experimentally induced change in both copies of a given gene in eukaryotic genome (diploid).

• One current approach to overcoming this difficulty is the use of targeted mutagenesis via homologous recombination.

• Using this technique, it is possible to replace an endogenous allele (copy) of a gene with one that has been genetically engineered.

Mammals Made to OrderMammals Made to Order

• In some instances, new, and sometimes novel, gene have been introduced into the organism.

• In other cases, a specific gene has been inactivated, then introduced into the organism in order to learn more about the normal role of the gene in embryonic development, tissue differentiation, the development of cancer, or in the functioning of the immune system. ---- knockout animals.

Fig. Mammals made to order: production of transgenic mice.

ES cells: embryonic stem cells (they are totipotent)

* c-abl: the cellular homolog of an oncogene from the Abelson murine leukemia virus

OncogenesOncogenes

• Oncogene – a gene that, in its nonmutant “cellular” form, is involved in the normal regulation of cell division.– When disrupted or altered, it is associated

with development of certain forms of cancer.

Fig. (Continued) Mammals made to order: production of transgenic mice.

a neomycin like substance

Concerns and Problems of Concerns and Problems of Transgenic AnimalsTransgenic Animals

• If the engineered gene is essential to embryonic development, homozygous offspring may die in uterus.

• The recipient (or host) organism plays an important role, not yet fully understood, in the successful incorporation of the donor cells into the developing embryo and subsequent germ-line transmission.

Concerns and Problems of Concerns and Problems of Transgenic AnimalsTransgenic Animals

• In the current methodology nonhomologous recombination is far more frequent than homologous recombination, and rigorous selection and careful analysis of cloned, transformed ES cells are necessary before they can be used as donors.

Gene TherapyGene Therapy

• The cells to be genetically altered can first be removed from the body.

• The genetic alteration is carried out in vitro.

• Altered cells having the desired genotype can be selected in the laboratory.

• Reintroduced the cells into the original donor or other suitable recipient

Uses of Recombinant DNA Uses of Recombinant DNA Technology in MedicineTechnology in Medicine

• Detection of a genetic disease– Enzymatic assays

– Gel electrophoresis of gene products

– Immunological methods

– Chromosomal analysis

– Restriction analysis

Restriction Fragment Length Polymorphisms (RRestriction Fragment Length Polymorphisms (RFLPs) Associated with Sickle Cell AnemiaFLPs) Associated with Sickle Cell Anemia

Fig. Simplified example of a DNA fingerprint resulting from the presence of variable numbers of tandem repeats (VNTRs).

Use of DNA Fingerprints in ForensicsUse of DNA Fingerprints in Forensics

Use of DNA Fingerprints in Use of DNA Fingerprints in Paternity DeterminationPaternity Determination

Identification of Codling Moth with Specific DNA Markers (1)

Identification of Codling Moth with Specific DNA Markers (2)

Uses of Recombinant DNA Uses of Recombinant DNA Technology in PharmaceuticsTechnology in Pharmaceutics

• A gene that codes for a particular peptide, along with an appropriate promoter and a sequence that instructs the cell to secrete the gene product, are inserted into an expression vector (e.g., phagegt11)

• Examples– Somatostatin– Insulin– Human growth hormone– Interferons– Opioid peptides– Thymosin– Tissue plasminogen activator– Urokinase – Hemophilia factor VIII

Uses of Recombinant DNA Uses of Recombinant DNA Technology in PharmaceuticsTechnology in Pharmaceutics

• If the gene producing the therapeutic product is of eukaryotic origin, special problems must be taken into consideration:– Eukaryotic promoters are not usually recognized

by bacterial RNA polymerases; hence the eukaryotic gene must be linked to a bacterial promoter.

– The mRNA transcribed from the eukaryotic gene may not be translatable on bacterial ribosomes.

Uses of Recombinant DNA Uses of Recombinant DNA Technology in PharmaceuticsTechnology in Pharmaceutics

– Introns may be present, and bacteria are unable to excise eukaryotic introns.

– The protein itself often must be processed and bacteria cannot recognize processing signals in eukaryotic gene products.

– Eukaryotic proteins may be recognized as foreign material by bacterial proteases and degraded.

Uses of Recombinant DNA Uses of Recombinant DNA Technology in PharmaceuticsTechnology in Pharmaceutics

• To solve the problems that may encounter by using prokaryotic cells– Use of a yeast artificial chromosome (YAC)– Use of transgenic animals (primarily sheep

and cows)• Vaccine production, e.g. gp120 of HIV surface

antigen• Hemophilia factor IX• -1-antitrypsin (causes emphysema)

““Dolly”- the First Cloned AnimalDolly”- the First Cloned Animal

• The first successful cloning of a lamb from a cell (a fibroblast) taken from the udder of an adult sheep.– growth the fibroblast cells in

culture– remove the nucleus of the egg– transplant the nucleus of the

fibroblast cell into the enucleated sheep eggs

– Implant into the uterus of a female sheep

Problems Associated with Problems Associated with Cloning AnimalsCloning Animals

• Many of the cloned animals have been – grossly abnormal

– failed to survive

– exhibited significant growth abnormalities

– aged prematurely

– developed degenerative diseases such as arthritis at an unusually age

Transgenic PigsTransgenic Pigs

• Cloned pigs were genetically engineered as potential organ donors to humans

• The alpha 1,3 galactosyl transferase gene (GATA1), a gene causes the human immune system to reject pig organs, was “knocked out” in these animals

• Knocked out the GATA1 gene is not likely to eliminate the problem of tissue and organ rejection

To Produce Other Medical To Produce Other Medical Important SubstancesImportant Substances

• Recombinant vaccines– Hepatitis B virus– AIDs – HIV virus

• Recombinant tissue plasminogen activator (TPA) – for treating heart attack

• Interleukin-2 and -interferon – for cancer treatment

• Human growth hormone (hGH) – for treating hypopituitary dwarfism

Supportive Therapy and Supportive Therapy and Gene TherapyGene Therapy

• Supportive (replacement) therapy: the malfunctioning cells are replaced with normal cells.e.g. bone-marrow transplantation

• Gene therapy: restitution of the normal gene in vivo.e.g. genetic diseases (i.e. cancer); severe

combined immunodeficiency (SCID), familial hypercholesterolemia

A Gene Therapy TrialA Gene Therapy Trial• Severe combined immunodeficiency (SCID) is caused by

a defect in the gene which produces the enzyme adenosine deaminase (ADA).

• In individuals who lack this enzyme, the nucleotide 2’-deoxyadnosine accumulates in the bloodstream, killing both the B and T lymphocytes of the immune system.

A Gene Therapy TrialA Gene Therapy Trial

• Gene therapy – Removed T lymphocytes from the SCID victim– Mixed with a genetically engineered retrovirus

containing a normal copy of the ADA gene– Verified the ADA gene was introduced into the

genome of the T cells and they were functioning– Transfused the cells into the patient– Serum ADA levels rose

• Since T cells are relatively short-lived, this gene therapy procedure must be repeated periodically.

Other Examples of Gene TherapyOther Examples of Gene Therapy

• Familial hypercholesterolemia– A defect in the gene that codes for the low density l

ipoprotein (LDL) receptor.

– A functional copy of the LDL receptor gene was introduced into the separated liver cell by modified virus.

– Reintroduce the cells to the liver

• Vascular endothelial growth factor (vegf) stimulates growth of collateral blood vessels around the area of an arterial blockage.

The Problems to Be Solved The Problems to Be Solved before Gene Therapybefore Gene Therapy

• It is not possible to remove certain types of cells from the body in order to genetically to modify them, nor can all types of cell be grown successfully in culture.

• There are major problems inherent in attempting to introduce foreign DNA into specific types of human cells in vivo.– viral vector infect only certain type of cells in vivo– lack selectivity of infection– inset their DNA at random locations within the human

genome

A Successful Gene TherapyA Successful Gene Therapy

• The normal gene must reach the tissue which the gene is normally expressed, enter the cell.

• The introduced normal gene undergo homologous recombination with the defective gene

• The gene is expressed at an appropriate time, and at appropriate level.

Uses of Recombinant DNA Uses of Recombinant DNA Technology in AgricultureTechnology in Agriculture

• Plants– Fixing nitrogen– Improving photosynthesis– Providing resistance to pests, pathogens and

herbicides• e.g. Bacillus thuringiensis endotoxin gene

– Increasing resistance to frost, drought and increased salinity

• Animals– Reach maturity more rapidly– Produce more milk or leaner meat– Produce pharmaceuticals

Fig. A transgenic tobacco plant which has been transformed with a firefly lucieferase gene.

Agrobacterium tumefaciensTumor inducing (Ti) plasmid

Effect of an extra growth Effect of an extra growth hormone gene in micehormone gene in mice

• Transgenic mouse (left)

carries a gene for rat

growth hormone

• Normal mouse (right)

Other Commercial and Other Commercial and Industrial Applications Industrial Applications

• Fermentation of cellulose or even plastic waste

• Fermentation at high temperature• Oil-metabolizing genes insert into a marine

bacterium• Degradation of other toxic products in the

environment• Conversion waste into usable food for

animals or humans

Social and Ethical Issues Social and Ethical Issues

• Whether scientists are “playing God” by altering the genomes of living organisms, thereby altering their evolutionary future?

• What would happen if some of these recombinant plants cross-pollinate with their wild relatives?

• Are certain genetically engineered foods safe?

• Are genetic engineering legal?

Genetic Testing Genetic Testing

• Who should have access to the information derived from genetic tests?– Only the individual?

– His or her spouse?

– Parents?

– Children?

– Siblings?

– His or her physician, employer, or insurance company?

Gene Therapy Gene Therapy

• What types of traits should be altered, who should decide?

• Do we really know what is best for the future of our species?

• Who will access to gene therapy?– Will this be reserved for the wealthy?

– Will it be covered by our health insurance?

What about human cloning or What about human cloning or transgenic humans ?transgenic humans ?