Aspects of genetic Engineering.

What is genetic engineering?

y Genetic engineering is the manipulations or the

change of DNA in a cell, so as to change the protein which that cell synthesises. y Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype. y It involves taking a piece DNA from one organism and placing it in another . y The new DNA formed is called recombinant DNA.

Types of enzymes involved in genetic engineering

Restriction endonucleases

Types of enzymes involved in genetic engineering

y DNA Ligases- these enzymes stick lengths of dna

together y DNA Helicase- Unwinds dna for dna replication or transcription y DNA polymerase-Adds nucleotides to make dna in dna replication y RNA polymerase- Adds nucleotides to make mRNA in transcription.

Genetic engineering
y To create new recombinant DNA, you must be able to:

Locate a specific gene in the donor 2. Isolate this gene as a piece of donor DNA 3. Modify the DNA in a highly selective way. 4. Transfer the modified donor DNA into a host cell in such a way that the gene with be expressed strongly enough to be of practical use.
1.

Locating and isolating gene

y Techniques include: 1.

Using genetic probes- In this at least part of the DNA base sequence in the gene must be known. The probe consist of a single strand of DNA that contains the known sequence of bases. It is labeled with a radioactive or fluorescent marker. Given the right conditions, the bases in the genetic probe combine with the complementary bases on the donor DNA, revealing the position of the gene. Once located, the gene can now be extracted.

Genetic probe

Locating and isolating gene

y Following the use of a genetic probe a shotgun

approach can be used y Shotgun approach: this involves the chopping up of DNA with restriction enzymes and searching for the piece with the required gene. y The piece is searched for by separated the fragments using gel electrophoresis. y This allows the fluorescent gene to be shown up and easily identified.

Gel electrophoresis

Locating and isolating gene

2. Reverse transcriptase: Cell that produce large amounts of a particular polypeptide will have large amounts of mRNA for the polypeptide. If this mRNA can be isolated, then its complimentary DNA (cDNA) can be synthesised. To do this, a special enzyme called reverse transcriptase is mixed with the mRNA together with free nucleotides, and as a result a complimentary single- stranded DNA is formed. The mRNA is the removed. The enzyme DNA polymerase is added together with more nucleotide and as a result a double- stranded DNA is formed. Normally

Modification and transferring

y Modifying a gene in a highly selective way may include

addition of: 1. sticky ends or 2. blunt ends (depends on which enzyme you use) y Transferring include the use of: 1. vector plasmid 2. Micro-injection 3. Electroporation 4. Lisosome transfer

Micro-injection & Electroporation

Insulin production
y Insulin is a (protein) hormone which is produced in y y y y y

the pancreas from cells call the Islets of langerhans. It converts excess glucose in to blood into glycogen and stored in the liver and muscles. Failure to produce insulin results in a disease called diabetes mellitus. People who are diabetic require regular injections of insulin or else they will go in coma or even die. Where does the insulin comes from? Lets see ..

Insulin production
y The first task was to isolate the gene coding for human y y

y y

insulin. This was done by first extracting mRNA from beta cell in the pancreas that produces insulin. The mRNA was then incubated with reverse transcriptase which came from a special group of viruses called retro viruses. This enzyme caused dna to be made from the mRNA. First single-stranded dna was formed which was then converted into double-stands using DNA polymerase.

Insulin production
y In order for this new insulin gene to be able to stick y y y y

onto other dna, sticky ends were added. This was done by adding lengths of single-stranded guanine nucleotides. In order to get the human insulin gene into a bacterium, a vector plasmid was used. A plasmid is small circular piece of dna which is found in bacteria separated from it normal dna. Plasmids are able to insert themselves into bacteria.

Insulin production
y To get the plasmids, the bacteria s cell wall was

dissolved using enzymes. The mixture was then centrifuged to separate the cell wall from the plasmid. y The plasmid were extracted and cut open with restriction enzymes. Once again sticky ends were added but this time cytosine was added. y The cut plasmid and the human dna were mixed with DNA ligase which joined them so that the plasmid now contain the human dna coding for insulin.

Insulin production
y The recombinant plasmids were mixed with the

bacteria Escherichia coli. y The plasmid entered the bacteria. The bacteria were then cultured, and allowed secrete insulin. y The insulin is extracted, purified and sold for to use of people with diabetes.

Gene therapy
y Gene therapy involves the addition of healthy, working copy of faulty genes into the appropriate cells in the body. y There are two type: 1. Somatic 2. Germ line y Somatic cell gene therapy involves the modification of a cell in a patient s body apart from the reproductive cells. y For example treatment of cystic fibrosis in lungs cells. y The genetic alteration is not passed to the patient s offspring. y Somatic cell gene therapy is the only form that is permitted in the UK.

Gene therapy
y Germ line gene therapy involves genetically modifying

a fertilized egg and therefore affect not only the individual that develops, but also their offspring and successive generation. y This form is currently banned in the UK. y This is because of serious ethical and health issues.

Gene therapy and cystic fibrosis.

y Cystic fibrosis (CF) is and inherited disease of your mucus and sweat gland. It affects mostly your lungs, pancreas, liver, intestines, sinuses, and sex organs. y Normally, mucus is watery. It keeps the lining of certain organs moist and prevents them from drying out or getting infected. y In CF a recessive gene causes mucus to become thick and sticky. y The normal dominant allele results in the production a membrane protein called cystic fibrosis transmembrane protein (CFTP). It is a carrier protein which transport chloride ions out of cells into the mucus.

Gene therapy and cystic fibrosis

y As chloride ions leave the cells, water leaves by osmosis

making the mucus watery. y In CF the chloride channels do not work, which results in a thick, sticky mucus. y This blocks the passage and allows growth of bacteria and results in infection. y A person with CF receives nasal spray with lososomes containing good gene which enters the person s lungs cell, and temporarily allow for chloride channel to function and allowing some relief.

Benefits of gene therapy

y The national cancer institute has successfully reengineer immune cells, called lymphocyte, to target cancer cell in patients with cancer. y Gene therapy is used to threat patients with cystic fibrosis. y Myeloid disorder is a disease affection the proper functioning of your bone marrow. Gene therapy can cure diseases of the myeloid system. y New gene therapy approach repairs errors in messenger RNA derived from defective genes. y Gene therapy is used to threat children with X-SCID (severe combined immunodeficiency ) or the `bubble boy disease.

Hazards of gene therapy

y Immune response- Anytime a foreign object is introduced into human tissue, the immune system is designed to attack the invader. This risk of stimulating the immune system in this way is always a risk. This can lead to infection and cause more problems. y The gene can also be over expressed causing damage to the patient. y In gene therapy, sometimes viral vectors are used. There in rish that once inside the patient, the viral vector may recover and cause diseases y Gene therapy can lead to mutations and hence cancer, should the gene be delivered to the wrong place. y Further more the immune system s enhanced response to invaders makes it difficult for gene therapy to be repeated in patients.

Hazards of gene therapy

y In germ-line gene therapy, modification is done at the zygote state. There is strong ethical issues because people believe that no one has the right to change the genetic composition of a human being. If allowed people will want to dictate what features there child has. People will want to make perfect beings or super beings. y Biologically, germ-line therapy is potentially dangerous because we know very little about how genes function in the embryo stage. Tampering with genes in the zygote could have effects that might apparent affect the child of affect off-springs later on in life.