Gene Technology

Question 1

What is genetic engineering/ genetic modification?

Answer 1

The insertion of genes from one organism into the genetic material of another organism or changing the genetic material of an organism

Question 2

What is recombinant DNA?

Answer 2

New DNA produced by genetic engineering that combines genes from the DNA of one organism with the DNA of another organism

Question 3

What are the most widely used genetically engineered organisms?

Answer 3

Bacteria

Question 4

How can artificial copies of a desired gene be made using mRNA?

Answer 4

An mRNA molecules transcribed from the gene is used to produce the correct DNA sequence. This uses the enzyme reverse transcriptase and effectively reverses the transcription process to produce complementary DNA (cDNA). This acts as an artificial gene

Question 5

What is reverse transcriptase?

Answer 5

The enzyme used to make artificial copies of a desired gene by taking an mRNA molecule transcribed from the gene and using it to produce the correct DNA sequence

Question 6

How can artificial copies of a desired gene be made using restriction endonucleases?

Answer 6

Restriction endonucleases are used to cut up DNA strands into small pieces that can be handled more easily. Each type of endonuclease will only cut DNA at specific (restriction) sites within a particular DNA sequence. Some restriction endonucleases can cut the DNA strands in a way that leaves a few base pairs longer on one than the other forming a sticky end. Sticky ends make it easier to attach a new piece of DNA to them. Sticky ends will only join up to other compatible sticky ends

Question 7

What do you do after getting your desired gene in genetic engineering?

Answer 7

Integrate the new gene into the vector. Plasmids are frequently used as vectors to carry the DNA into a host bacterial cell. DNA ligases are used as genetic glue to join pieces of DNA together. This is how the desired DNA is inserted into another piece or DNA (the vector) that will carry it to the host cell. Once the plasmid is incorporated into the host nucleus it forms part of the new recombinant DNA of the genetically engineered or transformed organism. However in a bacterial host cell it normally remains as an autonomous plasmid capable of independent replication

Question 8

How do scientists identify the organisms in which transformation has taken place?

Answer 8

Scientists transfer specual marker genes alongside the desired DNA. Thesse genes are dependent on a particular nhtruent or cause organisms to fluoresce in UV light

Question 9

Why aren’t marker genes for antibiotic resistance any more?

Answer 9

People are concerned that if they spread into the environment they would carey genes for antibiotic resistance with them which would make it harder to treat bacterial infections

Question 10

What is replica plating?

Answer 10

A process to identify recombinant cells. It involves growing identical patterns of bacterial colonies on plates with different media. It allows the identification of colonies that can survive a particular antibiotic or which can’t survive without a particular nutrient. These are the bacteria which have been genetically modified

Question 11

What are the four stages of replica plating?

Answer 11

1) the bacteria containing the desired gene also contains a marker gene that makes them need a specific amino acid in the nutrient medium in order to grow. Bacteria, including some that have been genetically engineeted are grown on a master plate on a complete medium
2) the master plate is inverted and pressed on a sterile velvet surface, leaving an imprint of the colonies. The master plate is saved
3) a second plate (the replica plate) is inverted and pressed against the velvet surface to pick up and imprint of the colonies. The medium of the replics plate lacks the specific amino acid required by the marker gene. The replica plate is incubated. The colonies that grow have not been genetically modified as they can synthesise the missing amino acids
4) the replica plate is compared to the master plate, so genetically modified colonies can be identified and grown on

Question 12

What do successful vectors do?

Answer 12

• target the right cell
• ensures that the desired gene is incorporated into the host cell genetic material so it can be activated (transcribed and translated)
• does not have any adverse side effects

Question 13

What are gene guns?

Answer 13

They are used to produce recombinant DNA by shooting the desired DNA into the cell at high speed on minute gold or tungsten pellets

Question 14

Whaat type of viruses are used as vectors?

Answer 14

Harmless viruses that are engineered to carry a desirable gene. They the infect the animals cells

Question 15

What is liposome wrapping?

Answer 15

A technique for producing recombinant DNA that involves wrapping the gene to be inserted in liposomes which fuse with the cell membrane and can pass through it to deliver the DNA into the cytoplasm

Question 16

What is microinjection? (DNA injection)

Answer 16

A technique for producing recombinant DNA. DNA is injected into a cell through a very fine micropipette. This is manipulated using a micromanipulator, because the steadiest hand would tremble enough to destroy the cell. The method is quite hit and miss. Many cells have to be injected before one takes up the DNA succesfully but it is the method that has resulted in the most successful transgenic animals

Question 17

Why are viruses best at getting DNA into the nucleus than other methods but what is the problem with them?

Answer 17

They are better because inserting DNA into the genetic material of other organisms is how viruses work. However they can cause an immund response in some people

Question 18

What happens in a knockout organism and how is this done?

Answer 18

One or more genes are silenced ‘knocked out’ so they no longer function. This is done by inserting a new gene that is similar to the gene to be investigated but which makes the original DNA sequence impossible to so the gene is silenced and cannot make a protein. Knockout genes are usually accompanied by marker genes

Question 19

What can knockout organisms be used to do?

Answer 19

• identify the function of a gene.
• investigate disease and test potential treatments. Genes that are known to be non-functioning in human diseases can be knocked out to create animal models of the disease

Question 20

What will transgenic plants hopefully be able to help with?

Answer 20

• fighting disease

* providing enough food of the right nutritional value to feed the ever-growing world population

Question 21

How are genes usually introduced into a plant?

Answer 21

Using the bacterium Agrobacterium tumefaciens. It causes crown galls (tumours) in plants. It contains a plasmid called the Ti plasmid which transfers bacterial genetic information directly into the plant DNA. This is what normally causes the abnormal growth of the plant cells but modified plasmids can be used to carry beneficial genes into the plant genome.

Question 22

What processes produces whole new transgenic plants?

Answer 22

Plant cloning

Question 23

Why can GM plant tissue be used to grow a mass of new GM plants by tissue culture?

Answer 23

Because plant cells remain totipotent throughout life

Question 24

How does the cloning to make transgenic plants take place?

Answer 24

A small piece of GM plant tissue is grown on a gel medoun impregnaged with plant hormones. A mass of undifferentiated, genetically identical plant tissue is produced by mitosis. Transfereing the cells to another gel impregnated with different hormones ecourages the development of roots and shoots and large numbers of new identical GM cloned plants result

Question 25

What are the four stages of introducing genes to a plant?

Answer 25

1) the Ti plasmid is extracted from A. Tumefaciens 2) the gene to be carried to the plant is inserted into the Ti plasmid which is returned to the bacterium 3) the plant is infected with the modified bacterium and part of the Ti plasmid with the engineered gene becomes part of the plant chromosomes 4) A. Tumefaciens causes a tumour to develop on the plant. These plant cells contain the new gene. If tumour cells are taken and cultured, whole new plants can be grown from them, containing the new genes. These arr genetically engineered or transgenic plants

Question 26

What are some exaples of plants that have been genetically modified crops more resistant to adverse conditions?

Answer 26

* Flood - resistant rice: complete immersion in water destroys rice crops, but global warming is causing more severe flooding. Strains of GM rice have been developed that can withstand total immersion in flood water for up to 3 weeks and still produce around 80% of the normal yields. * pesticide resistance. Genetic modification of crop plants can produce plants that produce plants that make their own pesticide in their leaves. This means farmers don't need to use chemical pesticides which are expensive and can harm the environment * changing the nutrient value of plants: by genetically modifying crop plants scientists have the potential to change the balance of different chemicals in the crop. If the vitamin or protein content of food can be increased, or the balance of fats changed, the value of the food to the people that eat it can be greatly increased. It may even be possible to grow plants that contain vaccines against deadly diseases

Question 27

What do the main genetic modifications of soya beans include?

Answer 27

* herbicide resistance: the yield of soya beans is badly affected by competition with weeds so farmers are spraying herbicides to destroy weeds. However soya plants are also killed by many broad leated plant herbicides. So scientists genetically modify soya beans to be resistant to common weed killers * Fatty acud balance: oil from soya beans is widely used in food production but traditional soya oil contains relatively little oleic and stearic acid and relatively latge amounts of linoleic acid. Linoleic acid oxidises easily so soya tends to oxidise and go odd. Also linoleic acid is a polyunsaturated fatty acid while oleic acid is a monounsaturated fatty acid and studies have suggested that oleic acid is better for health than linoleuc acid. Scientists have produced GM soya beans that have a radically different balance of fatty acids with a lot of oleic acid and less linoleic acid

Question 28

What are transgenic animals?

Answer 28

Animals that have had their DNA modified using gene technology so that at least some of their cells contain recombinant DNA

Question 29

What doess the production of human proteins using transgenic animals involve?

Answer 29

The introduction of a copy of a human gene that codes for the desired protein into the genetic material of an egg of a different animal species. As well as the gene doe the specific protein there is also a protein, there is also a promoter sequence that makes sure the gene will only be expressed in the mammary gland of the lactating female. The fertilised and transgenic embryo is then inside a surrogate mother and the animal is born and grows to maturity. When the animal is mature and produces milk that milk is harvested and purified and the human protein extracted

Question 30

What has been one of the problems with producing transgenic animals?

Answer 30

Most of the transgenic sheep were destroyed when the company producing them hit financial difficulties

Question 31

Even if somatic gene therapy is successful if the patient has a child they will pass on their faulty alleles. What is a potential solution to this?

Answer 31

To alter the germ cells so that the faulty genes are no longer passed on. This could be done in the early embryo immedietly after in vitro fertilisation. The new individual would be free of the disease and would not risk passing it to their own offspring

Question 32

Why are people concerned about germ line research?

Answer 32

* no one is yet sure of the effect on an early embryo of such an invasive intervention and the impact might not become clear until years into the life of the individual. * it is difficult to know where to draw the line in genetic modifying as some people may modify non threatening fratures such as adult height ect.

Question 33

What are some of the issues often raised about gene technology?

Answer 33

* concern that if marker genes which include antibiotic resistance, or other transgenic traits got into wild plant populations there might be a build up of antibiotic resistance. This issue is increasingly being addressed by the use of different maker genes, e.g. fluroscence * concerns about infertile seeds. Scientists usually include marker genes in GM plants. These marker genes may include allels that ensure the plant is infertile so the plants cannot reproduce and cannot then spread into the environment. However it does also mean that fresh supplies of transgenic plant seeds have to be bought each year for planting. This is more of a concern where food crops are involved than when crops are being modified to use for medicine. The big worry is that this will disadvantage people in poorer countries * worries over ingesting alien DNA in GM food plants (although we eat many types of DNA and digest it) * environmental concerns about gene transfer from GM plants and animaks to wild species * strong objections to the use of animals and other organisms for scientific research as some people feek that genetic modification violates the rights of the modified organism * a real concern that gene technology will be the property of just a few companies in developed countries

Question 34

How do you produce DNA using reverse transcriptase?

Answer 34

* use a cell that synthesises large quantities of the specific protein so contain large quantities of mRNA * this mRNA is isolated, e.g. by breaking open the cell and centrifuging the contents * the enzyme reverse transcriptase and a supply of nucleotides are added. This results in the synthesis of a single strand of complementary DNA (cDNA) using the extracted mRNA as a template * another enzyme called ribonuclease H is then added. This breaks down the mRNA strand leaving a single stranded piece of DNA * DNA polymerase and DNA nucleotides are added. A complementary strand of DNA is produced. The result is a fragment of double stranded DNA that is a copy of the original hene wjthout the introns