GENE TECHNOLOGY The method by which we can change the DNA in a

cell, & so change the protein which that cell synthesises is called Genetic engineering or Gene technology.

One form of diabetes mellitus is caused by the inability of the pancreas to produce insulin. People with this disease need regular injections of insulin which was extracted from the pancreas of pigs or cattle. This was expensive and many people did not like the idea of using insulin from an animal.

Moreover insulin from pigs and cattle is not identical to human insulin and can have side-effects.

• The biotechnology companies then started work on the idea of inserting the gene for human insulin into a bacterium & then using this bacterium to make insulin.

1)Isolating the insulin gene---Insulin is a protein. mRNA carrying the code for making insulin was extracted from the cells in a human pancreas that synthesis insulin called β cells.

2)The mRNA was then incubated with an enzyme reverse transcriptase which comes from a special group of viruses called retroviruses .

3)This enzyme does something which not normally happen in human cells. It reverses transcription , causing the DNA to be made from RNA.

• Complementary DNA (cDNA ) molecules were formed from the mRNA from the pancreas cell.

• First single stranded molecules were formed which were then converted to double stranded DNA. These DNA molecules carried the code for making insulin. That is ,they were insulin genes.

• In order to enable these insulin genes to stick onto other DNA ,they were given “sticky ends” (because these bases are exposed or unpaired , they will attract and stick to the exposed bases of complimentary sticky ends.

• This was done by adding lengths of single stranded DNA made up of guanine nucleotide to each end ,using enzyme. The sticky ends should be cut with the same restriction endonuclease enzyme.

• Some restriction endouclease enzymes cut evenly so they do not have the sticky ends, instead they are called the blunt cuts as these cannot be paired with the others.

• Some of the restriction endonuclease enzymes cut uneven or staggered ends. These uneven ends produced sticky ends.

• Restriction endonuclease enzymes are found naturally in bacteria .

Inserting the gene into a vector• In order to get the human insulin gene into a

bacterium , a vector has to be used( vector is a molecule of DNA which is used to carry a foreign gene into a host cell ). The vector used here is plasmid.

• Plasmid is a small, circular piece of DNA which can be found in many bacteria. Plasmids are able to put themselves into the bacteria, so if we put a piece of human DNA in the plasmid, the plasmid can take it into a bacterium.

• To get the plasmids ,the bacteria containing them were treated with enzyme to dissolve their cell wall. They were then centifuged , so that relatively large bacterial chromosomes were separated from much smaller plasmids & cell debris.

• The circular DNA molecules making up the plasmid was then cut open using a restriction enzyme . Once again , sticky ends were added ,but this time the nucleotide used to make this single strands contained cytosine.

• The cut plasmid & the cut human DNA were mixed together & the C & G bases on their sticky ends paired up.

• The nucleotide backbones were linked using an enzyme called DNA ligase , so that the human insulin gene became a part of the plasmid. This created recombinant DNA.

Inserting the gene into the bacteria

• The plasmids were mixed with bacteria. In case of insulin the bacterium was E.coli.

• A small portion of bacteria took up plasmids containing the insulin gene . some of the bacteria take up the plasmid but most bacteria don’t. Those bacteria that take up the plasmid are said to be transformed.

• These bacteria were separated from the others using antibiotic resistance provided by another gene which was introduced at the same time as the human gene insulin gene.

• When the bacteria were treated with antibiotic, only the ones containing the resistance gene(the insulin gene) survived.

• The genetically modified bacteria are now cultured on a large scale . These secrete insulin, which is extracted , purified & sold for use by people with diabetes. It is called recombinant insulin because it is produced by organisms containing a combination of their own and human DNA.

• Plasmids often contain gene that confer resistance to antibiotics.

• There is some concern about using antibiotic resistance genes as markers in this way.

• If the antibiotic resistance genes spread to other bacteria, producing strains of pathogenic (disease causing) bacteria that we could not kill with antibiotics. In this case the risk is probably very small because the genetically modified bacteria are only grown in fermenters & not released in the wild.

• But now there are many different kinds of genetically modified bacteria around, some of which are used in situations where their genes might be passed on to other bacteria . If there were pathogens then we might end up with the diseases that are untreatable.

• Because of this risk, there is now much less use of antibiotic resistance genes in this way & the other ways have been developed in which the successfully transformed bacteria can be identified.

• One of them uses enzymes that produce fluorescent substances . These enzymes are obtained from jelly fish & they make a protein that fluoresces bright green in UV light

• The gene for the enzyme is inserted into the plasmid . Then we can identify the bacteria that have taken up the plasmid by shining the UV light onto them. The once that glow are genetically modified.

Promoters• Bacteria contain many different genes, which make

many different proteins but not all these genes are switched on at once. The bacteria only makes the proteins that are required in the conditions in which they are growing.

• Example– they only make the enzyme β galactosidase (which digest lactose) when they are growing in a medium that contains lactose.

• In a bacterial cell the expression of each gene is controlled by a region of DNA called the promoters.

• If there is no promoter present , the inserted gene will not be expressed . It is therefore essential to transfer a suitable promoter to the bacterium along with the inserted gene.

• Many genes have to be activated(switched on) before they can be expressed. This prevents protein being produced at a wrong time or at a wrong place. When the bacteria was first transformed to produce insulin , the insulin genes was inserted next to β galactosidase gene, so that they shared a promoter .

• The promoter switches the gene “ON” when the bacterium needs to metabolise lactose.

• If the bacteria were then grown in a medium containing lactose ,they synthesised both

β galactosidase & human insulin.

Electrophoresis• It is a technique which is used to separate DNA

fragments of different sizes. It is used for genetic profiling (genetic finger printing) & for investigating the sequence of bases in a particular length of DNA.

• A region of DNA that is known to vary between different people is chosen . These regions often contain variable numbers of repeated DNA sequence & are known as variable numbers tandem repeats (VNTR) sequence. Only identical twins share all their VNTR sequence.

• DNA can be extracted from almost anything that has come from person’s body eg a root of hair, saliva , tiny spot of blood.

• Usually the quantity of DNA is increased by using polymerase chain reaction (PCR).

• Some procedures in gene technology require large amount of same DNA but only a small sample may be available eg blood at crime scene.The polymerase chain reaction enables many identical copies of DNA to be made from a small sample

• DNA is then chopped into pieces using restriction endonuclease enzyme .

• Now the DNA is ready for electrophoresis.• For electrophoresis there is tank containing a

pure form of agar called Agarose gel. A direct current is passed continuously through this gel.

• The DNA fragments are attracted to anode (positively charged electrodes) & are slowly pulled through the gel by this attractive force.

• The smaller the fragments the faster they move.

• When the current is turned off , the gel contains DNA fragments that have ended up in different places.

• To make them visible they are first transferred very carefully ,onto absorbent paper which is placed on top of the gel.

• Now the paper is heated just enough to make the two strands in each DNA molecules separate from one another.

• Short sequences of single stranded DNA , called probes are added which have base sequence complementary to VNTR regions.

• These pair up with the separated DNA strands on the paper.

• The probes contain a radioactive phosphorus Isotop.

• If the paper is now placed on an X ray film , the radiation emitted by probes (which are stuck to DNA fragments) makes a film go dark.

• There is a pattern of dark strips on the film matching the position that the DNA fragments reached on the Agarose gel.

• Electrophoresis is also used when a gene or other section of DNA has its code read , a process known as sequencing.

Cystic Fibrosis

• It is a genetic disease in which abnormally thick mucus is produced in the lungs & the other parts of the body. A person with cystic fibrosis is very prone to bacterial infection in the lungs ,because it is difficult for the mucus to be removed, allowing bacteria to breed in it.

• A person with CF needs daily therapy to help them cough up this mucus.

• The thick mucus affects other parts of the body. The pancreatic duct may become blocked & the people with this disease often take pancreatic enzyme with mouth to help with digestion.

• Around 90% of men with CF are sterile because thick secretion blocks ducts in the reproductive system.

• CF is caused by a recessive allele of gene which codes for the transporter protein called CFTR (Transmembrane regulator) . This protein sits in the cell surface membrane of the cells in the alveoli & allows Cl ions to pass out of the cells.

• The recessive allele codes for a faulty version of this protein which does not act properly as a chloride ion transporter.

• Normally, the cells lining the airways & in the lungs pump out Cl- ions through the channel in the cell surface membrane formed by CFTR. This results in relatively high concentration of Cl- ions outside the cell.

• This reduces the water potential below that of the cytoplasm of the cells . So water moves out of the cells by osmosis. It mixes with the mucus there , making it thin enough for easy removal by sweeping movements of cilia.

• However, in someone with CF, this does’nt happen. Much less water moves out of the cell

So the mucus on their surface stays thick and sticky. The cilia or even tough coughing can’t remove it.

• In a normal cell the loss of Cl- ions pull water with it by osmosis. This keeps the surface moist and well lubricated. The CFTR protein forms channels for the Cl- ions in the cell surface membrane.

The CFTR GeneIn each of our cell we have two sets of chromosomes, so we

have two copies of CFTR gene. The CFTR gene is found in chromosome 7 and consists of about 250,000 bases .

• Mutation in this gene have produced several defective alleles. The commonest of these is deletion of three bases.

• The CFTR protein made using the code on this allele is therefore missing one amino acid. The machinery in the cell recognises that this is not the right protein & does not place it in the cell surface membrane.

• Because the faulty CFTR alleles is recessive , someone with one faulty allele and one normal allele is

able to make enough of the CFTR protein to remain healthy. They are a symptom free carrier of the disease.

• Each time two heterozygous people have a child , there is a one in 4 chances that their child will have the disease.

• Because it is caused by a single gene, cystic fibrosis could be used a good candidate for gene therapy.

• If the normal dominant allele could be inserted into the cells in the lungs ,the correct CFTR should be made. In theory there are no reasons why this should not happen, but in practice there have been major problems in getting the gene into the cell.

• In UK trials began in 1993. The normal allele was inserted into the liposomes (tiny balls of lipids) which were then sprayed as an aerosol into the noses of nine volunteers. This succeeded in introducing the gene into a few cells lining the nose but the effect only lasted for weeks because these cells have a very short natural lifespan

• Researchers in USA tried a different vector. In a trial involving several people with CF, they introduced the gene into a normally harmless virus and then used these to carry the gene into the passages of the gas exchange system.

• The genes did enter some cells there but some of the volunteers experienced unpleasant side -effects as a result of infection of virus. As a result the trials were stopped.

• To be used as a treatment the gene really needs to get into many cells throughout the respiratory system. But this has not been achieved so far.

• So a different approach has been taken . In some people with CF, the mutation in the gene has simply replaced one base with the another. This has created a “stop” codon in the middle of the gene. The gene is transcribed (that is the mRNA is made from it) in the normal way but translation

on the ribosomes stop when this codon is reached. This means that only a short length of the CFTR protein is made .

• A drug called PTC124 has been found to allow translation just keep on going across this stop codon ,so the entire protein is made , albeit with a wrong or missing amino acid in the middle of it.

• Clinical trials have shown hopeful signs that this may allow enough CFTR to be made to significantly relieve the symptoms of some people with CF.

• It is much easier to do than the “classic “ gene therapy , because it only involves the patient taking a pill every day.

The genetic counsellor• Where there is a couple that may have a child

with a genetic disease they may be referred to a genetic counsellor.

• Either of them have a genetic disease in their family.

• They belong to a group that is known to have high risk of genetic diseases( a group in which marriage between close relative is common)

• There is a history of recurrent miscarriages.• A woman is over 38(high risk of down syndrome)• High risk pregnancy

• The role of a counsellor is to provide the couple with information & to help them to understand the risk involved in them having a child.

• The counsellor will encourage them to make their own decision based on this information.

• Giving birth to a child disease is not uncommon because the recessive allele can be hidden in the family for many years. For eg- Cystic Fibrosis allele. 1 in 3300 babies are born with this disease . In America 1/8500. For African –Americans 1/17000. The . This is because most people who are carriers do not have partners who are also carriers. And even if they do then there is only a 1 in 4 chances that anyone of their children will have a disease.

• If the parents find themselves in this situation & the disease is serious they may ask if they should risk having another child. The counsellor can give them information which can help them to make the decision.

• In some cases a person might know that there have been instances of a genetic disease in their family & is worried that they might have the child with this disease, in this cases also counsellor can help to work out the possibility

• Sometimes , as in case of CF a tissue sample can be taken from the parent & is tested to find out which allele in the gene is present.

• This is called Genetic Screening.

• It may involve Pedigree analysis ,in which the information going back for several generation is used

• Example– A woman whose uncle had haemophiliamight want to know if there is any chance that she

could pass it on to her son. Woman who are carriers of haemophilia will be unaware unless they have had a haemphiliac son.

• Blood Testing in combination with Pedigree analysis shows how haemophilia tends to pass from one generation to another.

Genetic screening• Genetic Screening is the analysis of a person’s DNA to

check for the presence of a particular allele. This can be done in adults, in a fetus or embryo in uterus or in a newly formed embryo produced by in vitro fertilisation.

• In 1989 ,the first designer baby was created ,officially known as (PGD). The technique involved mixing the fathers sperm with the mothers oocytes in a dish – (normal IVF).

• At the eight- cell stage one of the cells from the tiny embryo was removed. The DNA in the cell was analysed and used to predict whether or not the embryo would have the genetic diseases for which both the parents were carriers.

• An embryo that was not carrying the allele that would cause the disease was chosen for implantation & the

Others were discarded.• It has been used to avoid the pregnancies in

which the baby would have had Duchenne muscular dytrophy , thalassaemia(formation of abnormal haemoglobin), haemophilia huntington’s disease.

• For some time , genetic testing embryo has been leaving prospective parents with very difficult choices to make if the embryo is found to have a genetic disease such as Down syndrome or cystic fibrosis . The decision about whether or not to have a termination is very difficult to make.

Somatic and germ cell gene therapy• Gene Therapy involves introducing a “correct”

allele into a person’s cell as a treatment for a genetic disease .

• So far all the attempts to do this in human body have involved placing the allele in the body cells known as “ somatic cells”. However another possibility would be to insert the allele into germ cell( cells involved in sexual reproduction) such as gametes or an early embryo.

• Example ---- a woman with CF could opt to try to conceive a baby using IVF. Eggs(oocytes) would be taken out from her in a normal way . Then the correct allele of the CFTR gene would be injected into an egg & this egg fertilised by a sperm to produce a zygote. It is illegal in humans.

• It has been successfully done in animals.• The problem that many people see with germ

cell therapy is that all the cells of a child are produced from this genetically engineered zygote,& therefore will all carry the gene that had been inserted .

• When the child grows up and produces eggs or sperms ,these gametes will also contain the allele & therefore it will be passed onto their children . This is called allele in germ line ,passed from generation to generation.

The ethics of genetic engineering• In 2004 UK law allowed an embryo to be chosen

that did not have an allele for a genetic disease ,& also one that did have the tissue type that would allow a successful transplant into a sick elder brother or sister, but it did not allow the addition of an allele to an egg, sperm or zygote.

• Some people believe that the law is allowing too much while the others think that it should allow more.

• Example– A foetus can now can now be screened for a genetic disease while in uterus using Amniocentesis. The parents may then decide to have a pregnancy terminated if the embryo is found to have a genetic disease.

The social & ethical implications of gene technology

• The applications of gene technology such as genetically modified food , gene therapy ,cloning of animals for farming raise many new ethical, moral and legal issues.

• The situation is being monitored & legal controls do exist such as the germ cell gene therapy & human cloning are both illegal in UK

• On may 1st 1998 Iceland became the first UK supermarket chain to remove all GM ingredients from its own –brand food

• On june 5th 1999the UK’s first farm-scale trial of GM crops were destroyed on the orders of the family trust which owned the land.

•On October 5th 1999Monsanto, one of the largest biotechnology company publically announced that it would stop developing “terminator seeds”. These GM seeds include sterility genes so that the farmers have to buy fresh seeds every year. Some social and ethical issues are considered below

Example Potential benefit Potential risk

Use of modified microorganisms to produce proteins

Cheaper production and wide availability of medically important proteins eg hormones and vaccines They may be purer than alternatives eg –human insulin

Some of the microorganisms used Eg- E.coli bacterium ,normally lives in the humans. Genetically modified bacteria could “escape “ from the laboratory & create a new strain of superbug with a risk to human health.

Example Potential benefits Potential risks

Gene TherapyEffective treatment of genetic diseases Eg CF ,relieving suffering and prolonging life

There is a concern that the techniques used for human genetic modification could eventually make possible “ designer babies “ with preselected genetic features eg height , IQ

Genetically modified Food

Cheaper food for richer countries & reduction in food shortage in poorer countries

Possible side effects for humans eg- allergic reaction . Also the danger of “genetic pollution “ with the spread of new genes from the modified crops to wild species “ the risk of new super weeds”

Ethical implication of genetic engineering

• Ethics are sets of standards by which a particular group of people agree to regulate their behaviour , distinguishing an acceptable from an unacceptable activity.

• Ethics change with time ,because people alter their view to their knowledge & experience.

• In 1974 genetic engineers worldwide accepted a self- imposed ban on some recombinant DNA experiments on the basis that they were too risky.

• The public was introduced to its first genetically engineered animal in 1982 & now transgenic animals are standard tools in research & in the production of

Pharmaceuticals.An EU committee set up to investigate ethical

aspects of biotechnology has as one of its aims that of improving public understanding & acceptance.

A survey published in 1993 by the open university found that four out of five people do not trust industry to tell the truth about genetic engineering and two out of three felt that industry takes shortcuts with safety.

Genetically modified (transgenic animals)

• Human genes have been introduced into farm animals such as sheep and goat to give transgenic animals, that can produce useful human proteins in their milk.

• The animals are milked and proteins are extracted & purified

• Examples of proteins which have been produced in this way include blood factor IX used to treat people with haemoplilia & ATT( alpha -1-antitrypsin) used as a part of treatment for emphysema, cystic fibrosis & other lung diseases.

Genetically modified plant crops

• Maize– has been modified to protect it against an insect pest , the corn borer. A gene from the bacterium bacillus thuringiensis is introduced which causes the maize to produce a protein which is toxic to the insect pest but is harmless to humans eating the maize.

• Flavr savr tomatoes—these tomatoes have been modified to slow down the ripening process , increasing the shelf life & reducing waste. This method works by blocking synthesis of an enzyme involved in ripening. An antisense gene is introduced which blocks expression of the enzyme gene.

• Soya bean plants---- These plants have been made herbicide(weedkiller) resistant. Bacterial genes are added which make the plant resistant to a particular herbicide. The crop can then be sprayed with herbicide to kill the weeds but the soya is not affected

Benefits and hazards of genetic engineering• Genetically modified organisms can be created

for specific use.• In the past such organisms were derived from

selective breeding or arose by chance of mutation. In contrast to organisms produced by selective breeding ,there is a tendency to see genetically engineered organisms as unnatural and unsafe.

• As a result of genetic engineering micro-organisms now produce many substances that they would not normally produce. Most of these modified micro-organisms are kept in industrial fermenters , provided that precautions are used, they cannot effect the general environment.

• A totally different set of problem emerges when genetically engineered organisms ,such as crop plants & organisms for the biological control of pests are intended for use in general environment.

• UK has good consideration for the risk & benefits of releasing GM organisms.

• Anybody conducting a field trip must access the risk to the environment. The department then decides whether to issue consent.

• A“fast track procedure “ is followed in which the consent is given fast for the experiments which are less hazardous.

• Much less information need to be given about these experiments & the permission to perform trials can be given in 30 days.

• A low hazard release might be a species that has no natural relatives in the UK & has been modified with a well known gene

• While a high risk release might be a native weed with a gene that had not been transferred before.

• David bishop & his colleagues released a GM virus onto cabbage to test their effect on the caterpillar of the cabbage looper moth.

• The virus, a baculovirus has been discovered in a moth that is not native to the UK, the alfalfa looper.

• The virus has been modified by adding a gene coding for one of the proteins of scorpion venom , which is lethal to insects but harmless to other animals

• The unmodified virus also killed the caterpillar, but the modified virus killed them more quickly reducing damage to the cabbages.

• The chances of virus spreading was also reduced since the modified virus killed caterpillar so rapidly that they produced very few viruses.

• The new field trial was to measure the effect of virus on the caterpillars of six other moths ,& to see how long the virus survived in the soil & on the cabbages

• The cabbage were grown in a fine netting enclosure to keep out birds & animals that might spread the virus.

• The result showed that non-target species were much less susceptible to the virus than were the cabbage looper caterpillars.

• Of the target caterpillars 80% died after 8 days in comparison with 1% of the non-target species.

• The test also showed that genetically engineered virus is being marketed as a biopesticide ,since it kills caterpillar faster than the unmodified virus.

Experiment relevant to herbicide-resistant plant

• The concern about genetically engineered crops are that1) The modified crop plants will become agricultural weeds

or will invade natural habitats.2) The introduced genes will be transferred by pollen to wild

relatives whose hybrid offsprings will become more invasive.

3) The introduced gene will be transferred by the pollen to unmodified plants growing on a farm with organic certification.

4) The modified plants will be a direct hazard to humans , domestic animals by being toxic or producing allergies.