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What’s all the fuss about?

Why did the announcement of the birth of Dolly the sheep in 1997 cause such an interest in the scientific community?

Why was there such a huge public interest in this scientific story?

Dolly was the first mammal ever to be cloned from an adult cell. She was produced by transferring the nucleus from an

adult differentiated cell into an enucleated fertilised egg by a process called nuclear transfer. From a scientific view

point her birth represented a remarkable breakthrough because it showed that the nucleus from a specialised cell could

lose its specificity and function like the nucleus of a newly fertilised egg.

To the media, she represented the dawn of an era in which human cloning became a possibility. Here we will explore

how Dolly came about and the implications of her being.

Background

An adult mammal, such as a sheep or a human, is made up of many trillions of cells. There are about two hundred

different types of cells including epithelial cells which line organs, bone cells, muscle cells, white blood cells which are

responsible for the immune system and nerve cells. Each of these cell types is derived from a single cell, the fertilised

egg or zygote.

Initially the zygote divides to form a ball of identical cells. Differences soon arise between various cells as they and their

descendants become set along a particular developmental pathway. Once a cell has differentiated, for instance into a

kidney cell, it remains a kidney cell and does not normally change into another type of cell. Differentiated cells do not

normally undergo further division but some, such as melanocytes or cartilage cells, do divide. When this happens, the

new cells are of the same type as the parent cell.

Once differentiation has occurred it tends to be stable and to be inherited through a number of cell generations, even

when cells are cultured in the laboratory. When certain tissues are wounded the cells that remain may undergo a partial

reversal of the processes of differentiation known as dedifferentiation. The dedifferentiated cells then divide to form

a mass of cells called a blastema. These cells then redifferentiate to participate in the regeneration of the tissue. In

most cases cells redifferentiate into the same cell type as those that gave rise to the blastema.

As cells differentiate they may gain or lose organelles and change in shape, ultrastructure and behaviour. Some cells

synthesise specific substances: for example mammary gland alveolar cells synthesise lactoglobulin and erythroid cells

make haemoglobin. The differences in composition and metabolism of tissues mainly result from from different genes

being switched on or off in different cells. Regulation of protein synthesis mainly by controlling the synthesis of mRNA

is the key to cytodifferentiation and each cell type synthesises mRNA from only a selection of its complement of genes.

Most differentiated cells continue to carry the organism’s full genetic code. Dolly could only be produced if a way could

be found to completely dedifferentiate an adult cell and make it “forget” its developmental past.

In 1975, a developmental biologist in Cambridge, John Gurdon, produced tadpoles by transferring nuclei from cultured,

adult amphibian keratinocytes into enucleated eggs (keratinocytes are epidermal skin cells that make keratin). Although

this involved differentiation into complex tissues and organs, the tadpoles did not develop into adults, leaving open the

question of whether a differentiated adult nucleus could be fully reprogrammed.


How Dolly was made

The key to producing Dolly came from understanding the intricacies of the cell cycle and choosing nuclei from cells in

the appropriate stage of the cell cycle as donor nuclei.

Cells were taken from the mammary gland of a 6-year-old Finn Dorset ewe and allowed to divide in culture. Starving

the cells caused them to exit the growth cycle and arrest in G0. This is a stage in the cell cycle during early interphase

before DNA synthesis takes place. Cells in G0 are described as quiescent, having entered a “resting” phase during

which protein synthesis is reduced to about one fifth its normal level.

The cytoplasm of the unfertilised egg is thought to contain factors that “reprogramme” nuclear DNA. These normally act

on sperm DNA but nuclear transfer experiments suggest they can also “reprogramme” DNA from somatic cells. Scientists

think that the nuclei of somatic cells that are resting rather than actively dividing are more amenable to being reprogrammed,

though they don’t know why.

Individual mammary gland cells in G0 were fused with unfertilised, enucleated eggs from Scottish Blackface ewes by

electric pulsing, which also activates the egg and starts the development process. Two hundred and seventy seven of

these ‘reconstructed eggs’, each now with a diploid nucleus from an adult animal were generated. Rather than implant

each of these into individual ewes, to reduce the number of animals required, the eggs were cultured for 6 days in the

oviducts of receptive ewes. Twenty nine of the eggs that appeared to have developed normally to the blastocyst stage

were implanted into 13 surrogate Scottish Blackface ewes. One became pregnant and delivered a live lamb, Dolly, 142

days later.

Signals from the cytoplasm of the egg “reprogrammed” the nucleus. It no longer expressed itself as the nucleus of a

mammary gland cell, but as a nucleus from a newly formed zygote.

DNA removedfrom egg

Mammary cellsgrown in culture

Culture embryo (7days)

Transfer of cell

Egg donors

Surrogate mother

Egg and cell fused

Giving birth to lamb (Dolly)

Recover blastocystsand transfer to

surrogate mother

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1. If a fully differentiated mammary gland cell were to divide, what type of cell would you expect to be produced?

2. Donor cells were “starved”, what does this term mean here and why do cell biologists use this technique?

3. Why did the egg containing the donated nucleus develop into a whole new animal rather than a ball of

mammary gland cells?

4. Normally penetration of the egg by the sperm cell signals the start of the developmental process. What can

replace this signal when no sperm are involved?

5a. During the set of experiments which resulted in Dolly being born, what percentage of ‘reconstructed eggs’

resulted in live lambs?

b. Comment on the commercial viability and ethics of this process.

6. Dolly is not an exact genetic copy of the Finn Dorset ewe whose mammary cell was used to create Dolly -

explain (hint - mitochondria).

KEY TERMS

nuclear transferzygotedifferentiation (cytodifferentiation)dedifferentiationmelanocyteblastemaenucleated eggquiescence


Proving Dolly was what she was claimed to be

Following her birth, there was some controversy over Dolly’s authenticity.

The donor cell was taken from a culture of mammary cells, which, it was

acknowledged, could contain both stem cells as well as terminally

differentiated epithelial cells. It was suggested, however, that a culture of

embryonic derived cells was used by mistake. Since the donor animal was

pregnant when cells were collected there was also a remote possibility that

cultures made from mammary tissue might also be contaminated with stray

circulating foetal cells. Two approaches, DNA fingerprinting and

microsatellite analysis were taken to quash any further speculation that

the donor nucleus might have come from foetal or embryonic tissue.

DNA fingerprint analysis

DNA from Dolly’s cells was compared with DNA from the cultured mammary

cells used in the nuclear transfer experiments. The results of the fingerprint

analysis are shown on the right:

Lanes 1,2,3,4 and 5 contain DNA derived from members of the same flock

of Finn Dorset ewes as Dolly’s donor.

4 minisatellite probes were used in this DNA fingerprint analysis.

Microsatellite analysis

DNA from Dolly, the mammary tissue from which the donor cell was derived

and cells from the same culture as the donor cell were all subjected to

microsatellite analysis using 10 different microsatellite markers. The

results are summarised in the table below:

MarMarMarMarMarkkkkkererererer Number of allellesNumber of allellesNumber of allellesNumber of allellesNumber of allelles Alleles prAlleles prAlleles prAlleles prAlleles present in cells fresent in cells fresent in cells fresent in cells fresent in cells fromomomomomfffffound in Finn Dorsetound in Finn Dorsetound in Finn Dorsetound in Finn Dorsetound in Finn Dorset DollDollDollDollDollyyyyy, mammar, mammar, mammar, mammar, mammary tissue andy tissue andy tissue andy tissue andy tissue and

populapopulapopulapopulapopulationtiontiontiontion culturculturculturculturcultured cells (size in base pairs)ed cells (size in base pairs)ed cells (size in base pairs)ed cells (size in base pairs)ed cells (size in base pairs)

TGLA53 7 151/151

SPS115 4 248/248

TGLA126 7 118/126

TGLA122 9 190/190

ETH3 4 104/106

ETH225 4 148/150

FCB11 6 124/126

MAF209 4 109/121

FCB128 5 112/112

Udder

C

ultur

e ce

ll

Doll

y 1 2 3 4 5


Further information in the Student Support Notes may help you answer the following questions

1. Does the DNA fingerprint supports or dispute the claim that Dolly is derived from the nucleus of a Finn Dorset

udder cell? Explain.

2. The DNA fingerprint analysis includes fingerprints of Finn Dorset sheep from the same flock as the donor

ewe. What do these show and why were they included?

3. Apart from the 3 experimental lanes, a special relationship exists between 2 other animals. Which lanes

contain this DNA and can you suggest how these animals are related to each other. Why include these

samples?

4. Why are two sizes given for each of the microsatellite probes? What does it mean if both of these are the

same?

5. How do the results from the microsatellite analysis dispute the allegation that Dolly was derived from a stray

fetal cell?

6. Which is the most polymorphic of the markers used in the microsatellite analysis of Dolly? Why is it better to

use markers which are highly polymorphic in this type of investigation?

KEY TERMS

DNA fingerprintingmicrosatellite analysis


Dolly has a little lamb

Dolly has produced normal lambs on mating with a Welsh Mountain ram.

Is Dolly ageing prematurely?

One concern raised was that Dolly might age prematurely because she was

derived from a somatic cell rather than a germ line cell. This concern comes

form the observation that as normal somatic cells age, changes take place in

their DNA.

At either end of every strand of DNA from eukaryotic cells there are special

sequences of DNA. These are called telomeres. In most vertebrates, telomeres are short G-rich sequences: 5’- TTAGGG-

3’ repeated hundreds or even thousands of times. At each cell division some of these repeated sequences are lost (on

average 40 - 200 base pairs are lost per division per cell). Their function is not entirely clear but telomeres are believed

to act as “protective caps” on the ends of chromosomes and are important for replication and the stability of the

chromosome.

The Hayflick limit states that there is a finite limit to the number of cell divisions a normal somatic cell can go through.

Normal human fibroblasts for example, stop dividing after 50 generations in vitro. This phenomenon is thought to be

due to the erosion of telomeres.

Telomeres are believed to shorten during DNA replication because normal DNA polymerase does not completely

replicate the ends of linear chromosomes. A special DNA polymerase enzyme called telomerase does a more complete

job. Unfortunately, telomerase is in short supply in normal somatic cells. Germ cells (cells which produce sperm and

eggs) have a much higher level of telomerase activity. This is thought to explain why their telomeres are much longer

than those found in somatic cells and may explain why telomere length does not normally change from one generation

to the next.

Telomeres usually measure 24 units in the cells of one year old ewes. When she was one year old Dolly’s telomeres

measured only 19 units, closer to the size expected of a seven year old ewe. In all other respects Dolly functioned and

appeared like a normal one year old ewe. Dolly is now 4 years old (year 2000) and has remained healthy.

Potential uses of nuclear transfer

• Animal breeders could use nuclear transfer to rapidly expand numbers of elite animals generated using traditional

breeding systems.

• Transgenic sheep and cattle are already used to produce valuable human pharmaceutical proteins in their milk and

transgenic pigs are being developed as donors of organs in human transplantation. Until now these animals have

been made by adding DNA by injection directly into eggs. This is inefficient because genes are not directly targetted

and there is no guarantee they will be expressed by the correct cells. New technologies make it possible to perform

much more sophisticated manipulations of DNA in cultured cells so genes are directly targetted. The nuclei from

cells with the desired modifications can then be used as donors in nuclear transfer experiments to generate genetically

modified cattle, sheep and pigs.

• Cloning could be used in conservation programmes. Blood samples, skin biopsies and other cell types could

be collected of endangered breeds of animals, grown in the laboratory, then frozen in liquid nitrogen for long

term storage prior to nuclear transfer as required. More information at http://roslin.ac.uk.


Ethical/moral issues and concerns

Points for consideration here include the following:

• Animal welfare - do the experimental procedures used in cloning research cause undue suffering

to the animals involved? How do we judge what is ‘undue’?

• Religious concerns - is it blasphemous to manipulate DNA and in some cases cross species

barriers?

• Unnatural - is modern biotechnology unnatural in that it goes against and interferes with Nature,

making it intrinsically wrong? Compare with traditional selective breeding methods.

Several concerns regarding animal biotechnology more generally

have been raised. It is important to decide which of these are justified

and which aren’t. Some of these concerns are explored below:

• Animals engineered to grow faster might produce unexpected and harmful results for those

who eat foods derived from such animals. This enhanced growth rate may also have welfare

implications for the animals. There is currently controversy over the use of milk and meat

produced in the USA by injecting cattle with growth hormone. It could be argued that cloning

is the safer option.

• Widespread use of cloning might narrow the gene pool and reduce genetic diversity, so producing

livestock which could be vulnerable to new diseases or other environmental threats. On the

other hand cloning could be used to amplify numbers of breeds on the brink of extinction.

• Animals engineered in biomedical research to be models of human diseases might escape and

introduce genetic disorders on to wild populations. Of course this is not a problem peculiar to

animal models produced by genetic modification techniques.

• Organs from genetically modified animals might transmit viral diseases if used in human

transplant surgery. Pig organs, for example, contain viral DNA which do not harm the pig, but

may affect humans. Others which affect pigs but not humans are also of concern here. Many

safety checks must be carried out before these organs are used on patients.

• Genetically modified animals might escape into the environment. Transgenic salmon designed

to grow at a faster rate might out compete normal salmon for scarce food if they escape into the

wild. Ecological problems can of course be caused by other human activities as well such as

burning fossil fuels.

Moral and ethical issues are dealt with fully in the BBSRC publication:

Ethics, Morality and Animal Biotechnology. by R. Straughan (available free of charge from the BBSRC, Polaris

House, North Star Avenue, Swindon SN2 1UH


1. Why was it important to show that Dolly could breed normally?

2. What are “telomeres” and why is it not surprising that when she was one year old Dolly’s telomeres were

about the same as that expected for a 7 year old ewe?

3. Why are the scientists working on Dolly not overly concerned by the revelations concerning Dolly’s telomeres?

4. Why might a farmer using embryos produced by nuclear transfer from cows in elite herds see a much bigger

jump in performance of the herd compared with a farmer using artificial insemination?

5. Discuss the moral and ethical issues raised by genetic modification and nuclear transfer techniques.

KEY TERMS

somatic cellgerm celltelomereDNA polymerasetransgenicgenetic diversity

Cystic fibrosis (CF) is the most common inherited disorder in children of white Caucasian descent, affecting about 1 in

2500 births in the UK. There are about 7,000 CF patients currently registered in the UK. CF is caused by a recessive

mutation in a gene which codes for a protein called the cystic fibrosis transmembrane conductance regulator (CFTR)

in the plasma membrane of epithelial cells as shown in Fig. 1 below. 1 in 26 of the population are carriers of the

disorder.

The CFTR gene codes for a 1480 amino acid protein. It is a large gene and so far more than 800 different mutations have

been described for it. Most of these mutations are very rare. The exception is a deletion of 3 nucleotides which codes

for a phenylalanine residue at position 508 of the CFTR protein sequence. It is called ∆F508 and accounts for about

70% of CF mutations worldwide. Testing for this mutation and 19 others identifies more than 90% of all mutations.

Some mutations result in no CFTR protein being synthesised at all; in others, such as the ∆F508 mutation, the structure

of the protein is incomplete and it becomes stuck in the endoplasmic reticulum, never reaching the plasma membrane.

In other mutations the protein reaches the membrane but it does not function normally.

The protein coded for by the CFTR gene controls the movement of chloride ions into and out of epithelial cells, such as

those lining the airways of the lung. In children born with CF these chloride channels are defective or absent. This

results in an abnormal concentration of chloride outside the cell, which in turn causes the mucus that coats epithelial

cells to become thicker and stickier than normal.

The mucus builds up in the pancreas, lungs, digestive tract and other organs and can harbour persistent and recurrent

infections. Daily physiotherapy, a special diet, antibiotics and other preventative treatments can improve the quality of

life for sufferers.

Those suffering from CF may only develop some conditions in adulthood; these include diabetes, cirrhosis of the liver

and infertility (especially in men).

Fig 1. Structure of CFTR channel indicating the predicted location of some CF-associated mutations (asterisks).

MSD, membrane-spanning domain; NBD, nucleotide-binding domain; R, R domain; Inside, intracellular; and Outside,

extracellular. The branched structures are sites where carbohydrate groups are attached. Reproduced with

permission from D. Sheppard, Bristol University.

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Activity: Answer the following questions:

1. The CF allele is inherited in a typical Mendelian fashion. Carriers of the disease show no symptoms. Using

appropriate symbols for the dominant and recessive forms of the CF gene, give the genotypes and phenotypes

for:

a) a CF sufferer;

b) a CF carrier;

c) a “normal” individual.

2. Draw a genetic diagram to show the genotypes of children born to parents both of whom are CF carriers.

What proportion of the children will be carriers for the disease?

3. 54 million people in the UK are of white european origin. Using the information given above calculate:

a) the number of CF carriers in the UK;

b) the chance of two carriers getting married.

4. Boys and girls have an equal chance of getting CF, using all the information you now have, decide which of

the following terms best describes CF?

sex linked

autosomal recessive

autosomal dominant

5. a) How many nucleotides code for the amino acids found in the normal CFTR gene?

b) How many nucleotides code for the amino acids in the CFTR gene in sufferers carrying the ∆F508 mutation?

c) Describe how the polypeptide chain generated from this faulty gene differs from the normal product.

6. Explain how a couple tested for the ∆F508 mutation could still produce a child with CF.

Extra question:

If you were planning to start a family, would you wish to find out if you and your partner were carriers of CF?

How would the results affect your plans?

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The most common CF mutations are routinely tested for in many laboratories in the UK. Anyone who has CF in their

family is offered a test when planning a family. Both partners need to take the test.

The test involves taking a simple mouthwash. Cells are collected in a weak sugar solution rinsed around the mouth.

DNA is extracted from these cells and a diagnostic test using PCR (polymerase chain reaction) is carried out on the DNA

samples. If there is a family history for a particular mutation, only that mutation needs to be tested for, but in the partner

who does not have a family history up to 20 of the most common gene mutations may be tested for using this technique.

In the tests shown in Fig. 1 below seven individuals have been tested for four different CF mutations. There are two

tracks for each DNA sample. ∆F508 and 621+1 show up normally in the first track; G551D and G542X show up

normally in track two. A signal in the “wrong” track means that the individual carries the mutation and if the signal is

absent in the normal track then the sample is homozygous for the mutation.

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Fig. 1 The results from analysing 7 individuals for the 4 commonest cystic fibrosis mutations in North-West

England are shown. The test was developed by Zeneca Diagnostics in collaboration with the Molecular Genetics

Laboratory in the Department of Clinical Genetics Royal Manchester Children’s Hospital. Reproduced with

permission from Dr. M. Super.

1. Your task is to describe each of the individuals shown in Fig. 1 using the terms normal, heterozygote, homozygote

compound heterozygote (when two different mutations are present in the same individual) and the 4 different

mutations. The first one has been done for you.

Individual 1 is NORMAL.

Individual 2 ……………………………………………….

Individual 3 ……………………………………………….

Individual 4 ……………………………………………….

Individual 5 ……………………………………………….

Individual 6 ……………………………………………….

Individual 7 ……………………………………………….

2. Which individuals are

a) Normal b) Carriers c) Affected

3. Using appropriate symbols, draw a pedigree of the possible outcome if Simon (individual 3) and Louise

(individual 6) were to produce a family.

621+1G551DG542X

∆F508

1 2 1 2 1 2 1 2 1 2 1 2 1 2

Lane number

1 2 3 4 5 6 7

Individual

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ACTIVITY: Should Mollie and Carlos have a baby?

Before starting this activity collect as much information as you can about cystic fibrosis.

Suggested reading:

• Carlos and Mollie’s story Chapter 4 “Your Genes, Your Choices”. In “Exploring the issues raised by genetic

research” by Catherine Baker. AAAS. Available on the Internet at http://www.ornl.gov.

• “Genes and You: Teaching about genetics from a human perspective” by Gill Mullinar. Cross-curricular materials

for Key stage 4. Published by the Wellcome Trust.

• MRC Research update 3 “Cystic Fibrosis: the quest for a cure”.

• The MRC web site: http://www.hgu.mrc.ac.uk/Research

• Support notes in this pack.

• Class resources

Background

Mollie and Carlos need to decide whether or not to have prenatal testing for cystic fibrosis before trying for a baby.

Carlos had a brother who was constantly ill and died young from cystic fibrosis. He has already been tested and knows

he is a carrier of the disorder and does not want to see another child suffer as his brother did. He would like Mollie to

be tested before they go ahead and start a family. Mollie has no family history of cystic fibrosis and thinks they should

take a chance because they would love the baby whether or not it had cystic fibrosis. What should they do?

Activity

There are a number of issues to consider before Mollie and Carlos can decide which path to take. Your task is to

consider each of these then decide what you would do if you were Mollie or Carlos.

Use the following questions and ideas to help form your case:

• Find out what you can about CF (cystic fibrosis) – how it is caused; its symptoms; life expectancy of sufferers.

• How common is CF ?

• Carlos is a “carrier” for CF –explain.

• Boys and girls have an equal chance of getting CF – what does this tell you about the gene responsible?

• If Mollie decides to be tested for CF, what are the chances they will have an affected baby if

a) she is not a carrier ?

b) she is a carrier ?

• If they decide to go ahead and try for a baby, several prenatal tests are available, including alpha-fetoprotein test

(AFP), ultrasound, amniocentesis and chorionic villus sampling. Briefly describe each of these and place them in

order of risk to the foetus and usefulness (from greatest to least risk).

• Is it possible that Mollie and Carlos could still have a CF baby if the normal tests are negative?

• What are their choices?

• What is the likely outcome if a foetus is found to have 2 mutated CF genes?

• Other choices include pre-implantation diagnosis – what does this entail and what are the advantages and

disadvantages ?

• What would you do?

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ACTIVITY: Mollie and Carlos go for genetic testing

Mollie refused any prenatal testing and the couple decided to go ahead and have a family. Over the next few years they

had two normal, healthy sons, Hamish and Gordon. Their third child, Morag, was a daughter with cystic fibrosis. Soon

after Morag was born, Carlos and Mollie decided to take out life insurance policies for the whole family. The insurance

company insisted that the entire family underwent genetic testing for a range of genetic disorders including CF before

offering cover. This time Mollie agreed.

A small sample of blood was taken from each individual in the family. The white blood cells were isolated from the

samples and several genetic tests were carried out using microsatellite analysis. The results for one CF test is given

below:

INDIVIDUAL MARKER SIZE (bp)

Carlos 185/182

Mollie 185/182

Hamish 185/182

Gordon 185

Morag 182

Use the information above and in the rest of the pack to answer the following questions:

1. Complete the following table using the terms carrier, normal or CF:

INDIVIDUAL MARKER SIZE (bp) PHENOTYPE

Carlos 185/182

Mollie 185/182

Hamish 185/182

Gordon 185

Morag 182

2. Why were only white blood cells used for testing?

3. Describe the process used to produce the information in the tables.

4. Is CF a sex linked disorder? How does the information you have been given support your answer?

5. What is Carlos’ reaction likely to be when he is told about his sons’ results?

6. Describe the mutation detected in the CF test used here.

7. The insurance company has also seen the test results. Will the results affect the families wish to take out life

insurance?

8. 1 in 26 of the population are known to be carriers of CF.

(a) What are the chances of two carriers getting married?

(b) What are the chances of Hamish’s future wife being a CF carrier?

(c) What proportion of the offspring produced by two carriers will have cystic fibrosis?

(d) Why is the proportion given in answer (c) only used as a guideline when counselling prospective parents?

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New medicines and new approaches:GENE THERAPY

Genetic disorders such as cystic fibrosis, albinism, Huntington’s chorea, phenylketonuria, haemophilia and muscular

dystrophy result from mutations in the genetic code. The mutated gene gives out faulty instructions leading to the

production of an abnormal protein.

Phenylketonuria is usually tested for soon after birth and if detected this early it can be successfully treated by careful

control of diet. Unfortunately treatment is not usually this straightforward. One solution under investigation for genetic

disorders involving single gene defects is gene therapy, where the defective gene is compensated for by introducing a

normal one. This is a very attractive proposition because it tackles the cause of the disorder rather than the symptoms.

Every cell in the affected individual carries the faulty copy of the gene, but the gene only needs to be replaced in cells

which produce the protein encoded for by this gene and cause disease symptoms. In somatic cell gene therapy, a

normal copy of the gene is delivered to those cells so they can produce the normal protein. Germ line gene therapy is

much more controversial as it involves modifying a gamete, fertilised egg or embryo before the germline has split off

from the cells that will make the rest of the body, so any change made will be passed on to subsequent generations.

Germ line gene therapy is currently prohibited because of the ethical concerns it raises.

Before gene therapy can even be considered, the gene or genes responsible for the disorder need to be identified from

the estimated 70,000 to 140,000 genes present in the human genome. In 1989 the human gene responsible for cystic

fibrosis was identified on chromosome 7 and named CFTR.

The aim of gene therapy is to get normal copies of this gene into the epithelial cells lining the airways of the lungs. CF

is regarded as a good candidate for gene therapy because the cells affected line the lung passages so they can be treated

directly without being removed from the body. Scientists are working on a way of administering the normal gene using

the type of inhaler used commonly by asthmatics. This type of delivery is ideal since it causes the patient no discomfort.

Since the DNA is not integrated into the nuclear DNA, it is not replicated when the cell divides. This means that the

treatment has to be repeated every 2 to 3 months.

KEY TERMS

phenylketonuriahaemophiliamuscular dystrophyHuntington’s diseasegene therapysomatic cellgermlinebacterial plasmidvectorsadenovirusliposomeslipid bilayer

Human epithelial cell

Adenovirus

Cell surfacereceptor Nucleus

Viral and normalCFTR DNA

Normal CFTR proteinexpressed and correcting iondefect in plasma membrane

The normal form of the human CFTR gene has been isolated and inserted into a bacterial plasmid (circular piece of

DNA) and cloned to produce vast numbers of identical copies of the gene for experimental use. The next step is to find

a safe way to deliver this normal gene into the epithelial cells of CF sufferers. Two carrier systems, or vectors, have

been developed:

• The normal CFTR gene has been attached to the DNA in an adenovirus which has been treated to make it

completely safe to use. This is an example of a viral vector.

• liposomes can also be used as vectors for the CFTR gene. Liposomes are tiny hollow spheres made up of a

positively-charged lipid bilayer. They can be complexed with DNA and pass easily through cell membranes.

Fig.1 Adenovirus vector. The normal CFTR gene is inserted into the viruswhich infects the cell by binding to a cell surface receptopr. DNA enters thecell and is transported to the nucleus where the normal CFTR gene isexpressed and the normal protein produced.

Fig. 2 Positively charged liposomes combine with the negatively charged DNA to form a complex which fuses with the plasmamembrane and enters the cell. Inside the cell the DNA is released and transported to the nucleus where it is inserted into thecell’s DNA, so the normal protein can be produced.


Humanepithelial cell

Normal CFTR proteinexpressed and correctingion defect in plasmamembrane

Liposome spheremade up of apositivelycharged lipidbilayer

NormalCFTR gene

Liposome/DNA complex

nucleus


PROBLEM SOLVING ACTIVITIES

(Option 1)

1. How might the Human Genome Project be used to help sufferers of genetic disorders?

2. What is meant by the term “clone” as used in this passage?

3a) Explain why viruses are being used in the development of gene therapy.

b) Why is it important that the adenovirus be made safe before use?

c) Are there any disadvantages of using a viral delivery method?

4a) Explain why the composition of liposomes make them ideal vectors for gene therapy.

b) Give one reason why liposomes might be more popular than a virus for use as a vector.

5. Why should the type of gene therapy described here for CF be referred to as a treatment rather than a cure?

(Option 2)

You are the parents of a three year old son with CF. The disorder is being managed quite well with conventional treatment - drugs and

physiotherapy. There is still the constant worry of lung infections and hospitalisation. You are however very hopeful that your son

will grow up and lead as near a normal life as possible, his happiness and quality of life are very important to you.

You have discussed your concerns with your doctor and she has suggested the possibility of your son joining a gene therapy trial.

You are offered the chance of putting your son into a trial using either the liposome or the adenoviral vectors. Both vectors carry

the same corrected form of the CFTR gene. Would you wish your son to be part of a trial and if so which one would you choose?

Make an argument to persuade the doctors to enter your son in one of the trials - there are limited spaces available. You may wish

to consider the following points in your argument:

• your son’s age

• the current state of your son’s health

• whether or not you live close to the centre where the trials are being run

• how the therapy works

• the relative safety of the different vectors

• the results of other gene therapy trials

• the form treatment will take

• how long the trial will last


New medicines and new solutions:BIOPHARMACEUTICALS

Human pharmaceutical proteins currently in use include;

• human insulin for diabetes;

• human growth hormone for dwarfism;

• Factor VIII for haemophilia and

• Factor IX for haemophilia.

Several hundred other human proteins are currently at various stages of clinical evaluation, including;

• blood proteins;

• monoclonal antibodies;

• growth factor;

• soluble receptors and

• nutraceuticals.

Human pharmaceutical proteins are usually expensive to produce and demand outstrips supply. In the past the main source of many

of these proteins was cadavers and donated blood and there have been several problems associated with each of these (for example

transmission of hepatitis and HIV viruses and the agents responsible for Creutzfeldt-Jakob disease).

Genetic engineering has made it possible to clone protein-encoding genes. These recombinant genes can be expressed in a variety

of systems ranging from simple microbial systems such as bacteria, yeast and cultured mammalian cells to complex eukaryotic

systems such as transgenic plants and animals.

Most of the proteins pharmaceutical industries are interested in are secretory proteins which undergo complex post-translational

modifications as they pass through the secretory pathway in the cell. Often the protein will not function properly unless it has gone

through these modifications. Unfortunately for the genetic engineer, prokaryotic systems like bacteria cannot perform most of these

modifications. Yeast and higher plants can make some of them, but when the protein is intended for therapeutic use, a nearly perfect

match is required, so often only mammalian cells will do.

Figure 1 Shows the cost of producing raw material from which the pharmaceutical protein is extracted, assuming

40,000 litres are produced per annum.

0

20

40

60

80

100

120

plasma cell culture yeast transgenics

£ pe

r lit

re


Another option now available is the use of transgenic livestock.

Eukaryotic genes have regulatory sequences attached to them to determine which tissues they are expressed in. Genetic engineers

have identified those sequences which direct the expression of genes to the mammary gland. Any gene linked to these regulatory

sequences is expressed only in the mammary gland of a lactating animal. The pharmaceutical protein is produced in the milk and can

be purified from the milk.

Transgenic animals can be made by inserting recombinant DNA directly into one of the pronuclei of a fertilised egg. The embryo is

transferred to a foster mother, where it continues its development to birth.

The efficiency of producing transgenic livestock by this method depends on the species, but usually only about 3-5% of the animals

born carry the ‘transgene’. Only those animals which produce large amounts of the protein in their milk and can pass this ability onto

future generations will become founder animals for the large production flocks (see figure 2).

Figure 2: production of a herd of transgenic animals.

Production Diagram

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G1

G2

Transgenic founders

Male Females

Production unit


Choice of transgenic species

Figure 3 Livestock generation times (in months)

SpeciesSpeciesSpeciesSpeciesSpecies GoGoGoGoGo GoGoGoGoGo G1 birG1 birG1 birG1 birG1 birththththth G1G1G1G1G1 G2G2G2G2G2 G3G3G3G3G3BirBirBirBirBirththththth AdultAdultAdultAdultAdult (first milk)(first milk)(first milk)(first milk)(first milk) adultadultadultadultadult birbirbirbirbirththththth birbirbirbirbirththththth

Sheep 5 13 18 26 31 44Cow 9 23 32 46 55 78Pig 4 11 15 22 26 37Rabbit 1 6 7 - - -

Figure 4 Relative merits of species

SpeciesSpeciesSpeciesSpeciesSpecies VVVVVolume ofolume ofolume ofolume ofolume of milk milk milk milk milk Time to milkTime to milkTime to milkTime to milkTime to milk Ease ofEase ofEase ofEase ofEase of gener gener gener gener generaaaaatingtingtingtingting HealthHealthHealthHealthHealthprprprprproductionoductionoductionoductionoduction f f f f foundersoundersoundersoundersounders

Sheep +++ +++ ++ ++Cow ++++ ++ ++ ++Pig + +++ + +++Rabbit + ++++ ++++ +++

Figure 5 Protein produced in the milk of transgenic livestock

0 5 10 15 20 25 30

Expression level in g/litre

AAT

Fibrinogen

Antibiotics

Protein C

Antithrombin III

AAA- alpha-1-antitrypsin

Rec

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pro

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(Data for figures 1, 3, 4 and 5 kindly supplied by PPL Pharmaceuticals)


Activity

Using the information above answer the following questions:

Calcitonin is a peptide hormone secreted by the thyroid gland in mammals. It lowers the level of calcium in the blood

by reducing release of calcium from bone. A pharmaceutical company is investigating the possibility of producing large

quantities of calcitonin for the treatment of osteoporosis.

1. Use the information in figure 1 to answer the following questions:

i) Which is the most expensive option and what other reason is there for not choosing this option?

ii) The human calcitonin gene has been cloned. Why does the company not even consider the possibility of

using bacteria to produce the calcitonin. It would be much cheaper to use bacteria

iii) Compare the annual cost of producing 40,000 litres of raw material for calcitonin extraction from mammalian

cell culture with that from transgenic livestock.

iv) What hidden costs are there?

v) What do the genetic engineers have to do to the human calcitonin gene before microinjection if they want the

transgenic animal to produce human calcitonin in its milk?

2. Use the information in figure 2 to answer the following questions:

a) Why choose a male animal to found G2, the production flock?

b) What is the purpose of producing the females in G1?

3. Use figures 2, 3 and 4 to answer the following questions:

a) How long does it take to go from microinjection of the transgene to production of raw material in (1) cattle; (2)

sheep and (3) pigs?

b) What are the main (1) advantages, (2) disadvantages of choosing cattle to produce recombinant protein in

their milk?

c) A biotechnology company decides to produce flocks of transgenic sheep to produce human therapeutic

proteins, explain why they choose sheep rather than pigs or cattle.

KEY TERMS

HepatitisHIVCreutzfeldt-Jakob diseaseEukaryotic transgeneAlpha-1-antitrypsin

Activity (continued)

4. Use figure 5 to answer the following questions:

a) Complete the following table for annual production capacity:

Milk vMilk vMilk vMilk vMilk volume per yolume per yolume per yolume per yolume per yearearearearear Yield aYield aYield aYield aYield at 5g/l exprt 5g/l exprt 5g/l exprt 5g/l exprt 5g/l expressionessionessionessionession

Rabbits 8 litres

Sheep 300 litres

Cows 9,000 litres

b) Complete the following table for protein yield:

RRRRRecombinant precombinant precombinant precombinant precombinant proteinoteinoteinoteinotein Yield per y Yield per y Yield per y Yield per y Yield per year fear fear fear fear for for for for for flock oflock oflock oflock oflock of 50 e 50 e 50 e 50 e 50 ewwwwweseseseses

AAT

Antibiotics

Protein C

Fibrinogen

Antithrombin III

c) The annual market for AAT is estimated to be in the order of 1,000kg. The best mammalian cell culture system

working at maximum production levels can only produce approximately 10-20kg protein per year.

How many (1) mammalian cell culture systems (2) transgenic ewes, are required to meet the demand for AAT?

d) Explain how the technique used to produce Dolly the sheep could be applied to this technology.


Worksheet - COMPARATIVE MAPPINGWorksheet - COMPARATIVE MAPPINGWorksheet - COMPARATIVE MAPPINGWorksheet - COMPARATIVE MAPPINGWorksheet - COMPARATIVE MAPPING 11111

The arrangement of genes and other DNA markers is compared between species in Comparative genome mapping.

As early as 1915, the geneticist J.B.S Haldane reported that genes which are linked, or on the same chromosome, are

inherited together. From these early observations it was soon recognised that chromosomal segments are highly

conserved over long periods of time and any changes in gene order can be used, along with other evidence (such as the

fossil record), to chart the evolutionary history of a species.

As the Human Genome Project has unfolded there have been enormous advances in DNA technologies. The development

of PCR and automated sequencing especially have made it possible to construct genome maps with enough information

to make them useful for comparative studies.

Many mammalian genes have homologues in other animal groups. By comparing the amino acid sequences of the

proteins coded for by these genes, or the DNA sequences themselves, it has become clear that many of the 70,000 –

140,000 genes within the mammalian genome have been conserved over hundreds of millions of years of independent

evolution. Although we see a great variety in karyotypes, comparative mapping makes it clear that the order of genes

as well as the sequences of those genes is highly conserved in mammals.

Comparisons of human maps with those from other species provide clues into the evolutionary origin of regions of the

genome. Although interesting in its own right this may also provide new insights into the function of these regions. As

the initial stage of Human Genome Project nears its completion (with a completed DNA sequence expected by 2001),

this will become increasingly important as scientists attempt to determine the meaning of the DNA sequences generated.

Comparative genome maps made using genes and other DNA markers can be used to:

• predict the location of genes or markers in other species. If a new gene is mapped between two genes in

one species, its homologue is probably located between the same two genes in another species.

• identify candidate disease genes. Disease causing genes mapped in model species (such as the mouse)

have been used to locate the genes responsible for diseases such as Waardenburg Syndrome Type 1 and

neurofibromatosis in humans.

• characterise the genetic basis for complex genetic traits. Although comparative maps are valuable for

mapping single genes and identifying candidates for simple traits, they may be even more important for

analysing complex traits such as those responsible for epilepsy, diabetes and hypertension. It is impossible

to plan genetic crosses and control environmental factors in humans but this is possible in experimental

species such as the laboratory mouse and rat with the added bonus of being able to produce large numbers

of progeny. This approach is used to pinpoint regions of the genome important for complex traits. These

regions can then be thoroughly investigated.

INSTRUCTIONS

Genetic markers have been assigned to human and chicken chromosomes. By looking carefully at these markers:

Either:

Cut out human chromosomes 12 and 22 from the Chromosome Cut Out Sheet (H12 and H22) and paste the segments

onto the answer sheet to make the chicken chromosomes 1 and 18 (C1 and C18).

Or:

Cut out chicken chromosomes 1 and 18 from the Chromosome Cut Out Sheet (C1 and C18) and paste the segments

onto the answer sheet to make human chromosomes 12 and 22 (H12 and H22).

Remember to leave out any extra centromeres because each chromosome should only have one centromere.

Now answer the following questions:

1. Name the chromosomal mutations involved in the rearrangements you have just made.

2. Using the data provided is it possible to decide whether the chicken arrangement or the human arrangement is

closer to that of their common ancestor? If not what other data do you need?

3. Now use the additional information provided on Chromosome sheet 2 to decide whether the chromosomal

arrangement of the chicken or the human is more like the ancestral arrangement? Explain your answer.

4. You should now be able to complete the phylogenetic tree answer sheet.


KEY TERMS

linked geneshuman genome projectPCRautomated DNA sequencinggenome mapskaryotypes


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