CloningGene cloning – replication of donor genes in

bacterial or other host cells– clone – a group of cells, organisms, or genes that are

exact copies of each other– donor gene inserted into a bacterium is copied every

time the plasmid containing it replicates – genes can be cloned by growing genetically engineered bacteria

Controlling gene expression – genetic engineers want to be sure that only bacteria with donor gene replicate– to ensure this, a gene for antibiotic resistance is

attached to donor gene and antibiotic is used to kill all unwanted bacteria that do not have the donor gene

Polymerase Chain Reaction• cloning a gene through genetic engineering can be time-

consuming and requires an adequate DNA sample as starting material

• PCR technique allows researchers to amplify a tiny sample of DNA millions of times in a few hours

• DNA polymerase uses nucleotides and primers to replicate a DNA sequence in vitro, thereby producing two molecules

• Two strands of each molecule are then separated by heating and replicated again, so then there are four, double-stranded molecules

• After the next cycle of heating and replication there are eight molecules, and so on

• Number of molecules doubles with each cycle• PCR is useful in amplifying tiny samples of DNA ranging

from crime scenes to archaeological remains

• Cloning organisms – cloning sometimes occurs naturally (twins, asexual reproduction)

• organisms can be cloned artificially (sheep, rabbits, toads and other sexually reproducing animals have been cloned by dividing up an embryo and transplanting them into surrogate mothers)

Cloning of Dolly1.sheep cloned from a non-reproductive cell2. cell taken from udder of donor adult and cultured

in lab for 6 days3.unfertilized egg taken from another sheep –

nucleus removed4.egg without nucleus is fused with donor cell using

a spark of electricity5.embryo resulting from fusion of udder cell and egg

transferred into the uterus of a third sheep who acts as the surrogate mother

6.surrogate mother gives birth to lamb – lamb is genetically identical to sheep that donated udder cell

Human Genome Project• A project that involved mapping the entire human

genome – determined the order of all the bases in human DNA

• Outcomes of the HGP:

1. Determine how many individual genes we have and how they work.

2. Locating and determining the cause of genetic disorders.

3. Development of gene therapies to treat genetic disorders.

4. Comparing genetic makeup of human populations to determine ancestries and how humans have migrated and mixed their genes with other populations over time.

Practical Applications of DNA

Technology

• DNA technology is reshaping medicine and the pharmaceutical industry

– one obvious benefit of DNA technology and of the Human Genome Project (international cooperative venture established to sequence the complete human genome) is the identification of genes whose mutation is responsible for genetic diseases

Diagnosis of Disease and Genetic Screening (testing an individual for the presence or absence of a gene)– medical scientists currently use DNA

technology to diagnose hundreds of human genetic disorders and to detect other diseases such as HIV in blood or tissue samples (important to keep blood supply safe)

– this allows early disease detection and identification of carriers for potentially harmful recessive mutations – even before the onset of symptoms

• genes have been cloned for many genetic disorders including hemophilia, phenylketonuria, cystic fibrosis, and Duchenne muscular dystrophy – this allows direct detection of gene mutations– cloned normal gene can be used as a probe to

find the corresponding gene in cells being tested

• alleles for cystic fibrosis and Huntington’s disease can also be detected

there are advantages and disadvantages to genetic screening

• advantages include pre-natal diagnosis of genetic diseases (before the baby is born), ability to detect carriers of genetic diseases, confirmation of animal pedigrees (DNA testing required in dogs), resolution of immigration disputes

• disadvantages include ethical issues – i.e. question of abortion if fetus is found to have a genetic disorder

Human Gene Therapy – example: Cystic Fibrosis

• theoretically, it should be possible to replace or supplement defective genes with functional normal genes using recombinant DNA techniques

• CF is the most common inherited disease among northern Europeans and white North Americans

• due to a recessive gene located on chromosome 7• normal allele controls the production of a

membrane protein called cystic fibrosis transmembrane protein (CFTP) – essential for proper functioning of epithelial cells (normal epithelial mucus is watery) which traps dust and microorganisms – mucus moves particles up throat to be swallowed or blown out of nose

• CF patients have abnormally thick mucus – difficult to clear, clogs airways, breeding ground for bacteria

• lack of CFTP also causes growth of fibrous cysts in pancreas which interfere with delivery of digestive enzymes

• CF gene has been cloned allowing for identification of causes of disease, allowing for genetic screening, and possible gene therapy

• gene therapy for CF targets the lungs – gene for normal CFTP is isolated and inserted into a bacterial plasmid

• plasmid is introduced in bacterial cell for cloning by bacteria (i.e. multiple copies are made)

• plasmids are inserted into liposomes (sphere of lipid that can penetrate the cell surface membrane)

• liposomes are taken as nasal spray• liposomes enter cells of lung tissue; normal allele

is expressed• treatment only effective for a few weeks as

epithelial cells lining the respiratory tract are shed regularly

Pharmaceutical Products– DNA technology has been used to make

human insulin and growth hormone

Forensic uses – forensic labs can determine blood or tissue type from blood, small fragments of other tissue, or semen left at the scene of violent crimes– DNA fingerprinting can be used for

identification of criminals

Environmental uses

• genetically engineered microorganisms that can extract heavy metals from their environment

• genetically engineered microbes may be used in mining and in cleaning up mining waste

• bacterial strains have been developed to detoxify specific wastes found in spills and waste dumps

Agricultural uses of DNA technology Animal Husbandry – many farm animals are

treated with products made by recombinant DNA methods (examples include vaccines, antibodies, and growth hormones)

– some milk cows are injected with bovine growth hormone (BGH) made by E. coli, in order to raise milk production

– BGH also improves weight gain in beef cattle

Transgenic animals – animals that contain genes from another species have been developed for agricultural use (examples include beef and dairy cattle, hogs, sheep and several species of commercially raised fishes)– modified DNA can be introduced into diary cows so that

they produce human proteins – protein is produced in the milk – examples of medically important proteins that have been produced in transgenic mammals include:

• blood clotting Factor VIII to treat hemophilia• alpha-1- antitrypsin which helps protect the lungs

from damage during infections– rainbow trout and salmon that are given a foreign

growth hormone can reach in one year a size that usually requires 2 to 3 years of growth

Genetic engineering in plants

– plants have been genetically altered to receive herbicide resistance (several strains of cotton) – allows them to be resistant to herbicides used to kill weeds

– some crop plants are being engineered to resist infectious pathogens and pest insects – reduces need to apply chemical insecticides

• first genetically engineered fruits approved by the FDA for human consumption were tomatoes engineered with antisense genes that retard spoilage– researchers isolated gene responsible for

ripening– they prepared a gene who's template strand had

a base sequence complementary to the normal gene – an antisense version of the gene

– when spliced into the DNA of a tomato plant, the antisense gene is transcribed into RNA that is complementary to the ripening gene’s mRNA

– the antisense RNA binds to the normal mRNA, blocking the synthesis of the enzyme causing ripening and spoilage

Benefits and Possible Harmful Effects of Genetic Modification

• See handouts and Clegg pg. 127-128

Stem Cells• Cells that retain their ability to divide and

differentiate into various cell types

• Plants contain stem cells in their meristems (reason why a cutting can grow into a new plant)

• Embryonic stem cells are pluripotent – can form any type of cell in an organism or can form a complete organism

• Adult stem cells can divide to form new body tissue cells (i.e. blood stem cells)

Therapeutic Uses of Stem Cells• Embryonic stem cells are the most flexible

and can grow into any type of mature cell– Parkinson’s and Alzheimer’s Disease can be

potentially treated by implanting stem cells that could replace the damaged cells

Ethical Issues surrounding therapeutic cloning

• Therapeutic Cloning is the creation of an embryo to supply embryonic stem cells for medical use

– Raises issue of whether it is right or wrong to generate a new human embryo for medical research

• Two distinct forms of cloning:1. Reproductive cloning – making copies of entire

organism2. Therapeutic cloning – making copies of embryonic

stem cells• Opinions vary about whether both forms are

right/wrong or if one or the other is acceptable