aspects of genetic engineering

8/3/2019 Aspects of Genetic Engineering

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Aspects of geneticEngineering.


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What is genetic engineering?y

Genetic engineering is the manipulations or thechange of DNA in a cell, so as to change the proteinwhich that cell synthesises.

y Genetic engineering, also known as recombinant DNA

technology, means altering the genes in a livingorganism 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.


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Types of enzymes involved in genetic engineeringRestriction endonucleases


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Types of enzymes involved in

genetic engineeringy DNALigases- these enzymes stick lengths of dna

together

y DNA Helicase- Unwinds dna for dna replication ortranscription

y DNA polymerase-Adds nucleotides to make dna in dnareplication

y RNA polymerase-Adds nucleotides to make mRNA intranscription.


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

1. Locate a specific gene in the donor

2. Isolate this gene as a piece of donor DNA3. Modifythe DNA in a highly selective way.

4. Transfer the modified donor DNA into a host cell insuch a way that the gene with be expressed strongly

enough to be of practical use.


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Locating and isolating geney

Techniques include:1. Using genetic probes- In this at least part of the DNA

base sequence in the gene must be known. The probeconsist of a single strand of DNA that contains the

known sequence of bases. It is labeled with aradioactive or fluorescent marker. Given the rightconditions, the bases in the genetic probe combinewith the complementary bases on the donor DNA,revealing the position of the gene. Once located, thegene can now be extracted.


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Genetic probe


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Locating and isolating geney Following the use of a genetic probe a shotgun

approach can be used

y Shotgun approach: this involves the chopping up ofDNA with restriction enzymes and searching for thepiece 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.


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Gel electrophoresis


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Locating and isolating gene2. Reverse transcriptase: Cell that produce large amounts

of a particular polypeptide will have large amounts ofmRNA for the polypeptide. If this mRNA can beisolated, then its complimentary DNA (cDNA) canbe synthesised.

To do this, a special enzyme called reversetranscriptase is mixed with the mRNA together withfree nucleotides, and as a result a complimentarysingle- stranded DNA is formed.

The mRNA is the removed. The enzyme DNApolymerase is added together with more nucleotideand as a result a double- stranded DNA is formed.

Normally


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Modification and transferringy Modifying a gene in a highly selective way may include

addition of:

1. sticky ends or2. 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


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Micro-injection & Electroporation


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Insulin production

y Insulin is a (protein) hormonewhich is produced inthe pancreas from cells call the Islets of langerhans.

y It converts excess glucose in to blood into glycogen andstored in the liver and muscles.

y Failure to produce insulin results in a disease calleddiabetes mellitus.

y People who are diabetic require regular injections ofinsulin or else they will go in coma or even die.

yWhere does the insulin comes from?

y Lets see..


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

insulin.

yThis was done by first extracting mRNAfrom beta cellin the pancreas that produces insulin.

y The mRNA was then incubated with reversetranscriptasewhich came from a special group of

viruses called retro viruses.y This enzyme caused dna to be made from the mRNA.

y First single-stranded dna was formed which was thenconverted into double-stands using DNA polymerase.


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

onto other dna, sticky endswere added.

yThis was done by adding lengths of single-strandedguanine nucleotides.

y In order to get the human insulin gene into abacterium, avector plasmidwas used.

yAplasmid is small circular piece of dnawhich is foundin bacteria separated from it normal dna.

y Plasmids are able to insert themselves into bacteria.


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Insulin productiony To get the plasmids, the bacterias 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 wereadded but this time cytosinewas added.

y

The cut plasmid and the human dna were mixed withDNA ligasewhich joined them so that the plasmidnow contain the human dna coding for insulin.


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Insulin productiony The recombinant plasmidswere mixed with the

bacteria Escherichia coli.

y

The plasmid entered the bacteria. The bacteria werethen cultured, and allowed secrete insulin.

y The insulin is extracted, purified and sold for to use ofpeople with diabetes.


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Gene therapyy Gene therapyinvolves the addition of healthy, working

copy of faulty genes into the appropriate cells in the body.y There are two type:

1. Somatic2. Germ liney Somatic cell gene therapy involves the modification of a

cell in a patients body apart from the reproductive cells.y For example treatment ofcystic fibrosis in lungs cells.y The genetic alteration is not passed to the patients

offspring.y Somatic cell gene therapy is the only form that is

permitted in the UK.


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Gene therapyy Germ line gene therapy involves genetically modifying

a fertilized egg and therefore affect not only the

individual that develops, but also their offspring andsuccessive generation.

y This form is currently banned in the UK.

y This is because of serious ethical and health issues.


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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 certainorgans moist and prevents them from drying out or gettinginfected.

y In CF a recessive gene causes mucus to become thick andsticky.

y The normal dominant allele results in the production amembrane protein called cystic fibrosis trans-membrane protein (CFTP). It is a carrier protein whichtransport chloride ions out of cells into the mucus.


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Gene therapy and cystic fibrosisyAs chloride ions leave the cells,water leaves by osmosis

making the mucus watery.

y

In CF the chloride channels do not work, which resultsin a thick, sticky mucus.

y This blocks the passage and allows growth of bacteriaand results in infection.

yA person with CF receives nasal spray with lososomescontaining good gene which enters the persons lungscell, and temporarily allow for chloride channel tofunction and allowing some relief.


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Benefits of gene therapy

y The national cancer institute has successfully reengineerimmune cells, called lymphocyte, to target cancer cell inpatients with cancer.

y Gene therapy is used to threat patients with cystic fibrosis.

y Myeloid disorder is a disease affection the properfunctioning of your bone marrow. Gene therapy can curediseases of the myeloid system.

y New gene therapy approach repairs errors in messengerRNA derived from defective genes.

y Gene therapy is used to threat children with X-SCID(severe combined immunodeficiency ) or the `bubble boydisease.


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Hazards of gene therapyy Immune response-Anytime a foreign object is introduced

into human tissue, the immune system is designed toattack the invader. This risk of stimulating the immunesystem in this way is always a risk. This can lead toinfection and cause more problems.

y The gene can also be over expressed causing damage to thepatient.

y In gene therapy, sometimes viral vectors are used. There inrish that once inside the patient, the viralvector mayrecover 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 systems enhanced response toinvaders makes it difficult for gene therapyto be repeated

in patients.


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Hazards of gene therapyy In germ-line gene therapy, modification is done at the

zygote state. There is strong ethical issues because peoplebelieve that no one has the right to change the geneticcomposition of a human being. If allowed people will wantto dictate what features there child has. People will want tomake perfect beings or super beings.

y Biologically, germ-line therapy is potentially dangerous

because we know very little about how genes function inthe embryo stage. Tampering with genes in the zygotecould have effects that might apparent affect the child ofaffect off-springs later on in life.

aspects of genetic engineering

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