Topic 6 -

Gene technology-
i

8C I
-

Dr. Nihal Gabr

1/4/2023
8C genetic engineering
 Genetically modified organism……organisms that have genome ( DNA ) altered by genetic engineering

Scientists developed GMO …organisms have desirable features .

GM bacteria ……..insulin and growth hormone

Transgenic organism …….contain recombinant DNA GM Plant
Resistant to herbicides
Change Genes Beta carotene

Genetic engineering

By taking a gene from one organism and inserting it into

another organism .
Restriction
1. Gene
Reverse transcriptase
2. Many copies PCR
3. Vector ……
4. Join ligase
5. Promotor and gene marker
 The process of making a protein using genetic engineering has a number of stages

A Step 1 1. Obtain / synthesis the desired gene

A) reverse transcriptase B) restriction enzyme

Use RNA as a template to form Extracted from bacteria ….cut specific base
sequence ( restriction site / recognition site)
DNA
( reverse transcriptase produced by Cut double stranded DNA at a specific

retrovirus) recognition site leaving sticky ends

Mature mRNA with no introns +

easy to extract

1. A cell readily produce protein is

selected
2. They have large quantities of the
required mRNA which is extracted .
3. Reverse transcriptase is used to
make a single stranded cDNA
( complementary DNA) .
4. Then use DNA polymerase to make
another DNA strand , the double
stranded DNA is the required gene .
Step 1 1. Cut the gene of the desired protein using Restriction Endonuclease enzyme ( or obtain
the desired gene using Reverse transcriptase enzyme which acts on mRNA )

Step 2 Make many copies of the desired gene using pCR using
Taq DNA polymerase .

Step 3 Vector which is the plasmid removed from the bacteria and cut open using the same specific
restriction enzyme
Which leave sticky ends …which has complementary base sequence to sticky ends of the
gene .

Step 4 The amplified gene is mixed with the cut plasmid ( after adding promotor and gene
marker ) and DNA ligase link the sugar phosphate back bone and plasmid producing
a recombinant DNA ( ligase joins the gene to plasmid )
 Add a gene marker to the bacterial plasmid …gene coding for resistance to
Step 5
specific antibiotic / gene coding for a fluorescent protein ( GFP) / gene that
makes the bacterium dependent on a particular nutrients

Gene Gene of
Promotor
marker interest
Transcription factor / RNA polymerase
As the gene has to be inserted next to promoter
To identify the transformed bacteria
For TF to bind and initiate transcription.

. Identification of host cell that has successfully taken up the gene by using GENE MARKER

1. Make the fluorescent protein easily seen using UV light

Gene marker 2. Gene which causes the bacteria to become resistant t antibiotics
3. Makes the bacteria dependent on a particular nutrient .

Replica platting
We use replica platting to identify recombinant cells .
Growing bacteria on agar plates with different media .
This allows us to identify colonies that cant survive without a particular nutrient .
These are the bacteria that have been GM .
Transformed + non transformed

Culture the transformed bacteria over Nutrient medium.

Step 6
Allow large scale production inside a fermentor
 Identifiocation of the host cell has successfully taken up the gene of interest using
GENE MARKER ( transformed bacteria ) :

A GFP ( green fluorescence protein )

Easily seen using U.V light

B Making the bacteria dependent on a particular nutrient .

C Gene which cause the bacteria to become resistant to the antibiotics

Ampicillin
Tetracycline resistance
* C
resistance Normal plasmid of

Insulin gene had been inserted into the

E
bacteria

Gene of
interest plasmid at a point in the gene for
*
( insulin) tetracycline resistance V ~ A
Normal plasmid of
bacteria

1. Culture bacteria on an agar plate containing ampicillin antibiotic , where the plasmid containing
resistant gene to ampicillin will cause the bacteria with these plasmids to survive ….excluded C .
2. To identify the bacteria with recombinant DNA , add the bacteria using sterile velvet on an
agar plate containing tetracycline …so the transformed bacteria with recombinant DNA wont
grow on the plate containing tetracycline
Why use of antibiotic resistance gene as gene marker is not successful / not used anymore ?
1. Risk the spreading resistance to other bacteria …
2,. So antibiotics become no longer effective .

Explain the role of restriction enzyme in genetic engineering

1.The restriction enzyme

2.Used to cut gene out of animal DNA
Cut double stranded DNA at a specific recognition site leaving sticky ends
3.Make many copies of the desired gene using pCR using DNA polymerase .
4.Vector which is the plasmid removed from the bacteria and cut open using the same specific restriction
enzyme …5.to produce sticky ends .
6.The amplified gene is mixed with the cut plasmid ( after adding promotor and gene marker ) ..where hydrogen
bonds formed between bases at sticky ends.
7. and DNA ligase link the sugar phosphate back bone and plasmid producing a recombinant DNA ( ligase
joins the gene to plasmid )

Explain how cells / organism can be genetically modified ?

1.Use restriction enzyme
2.Used to cut gene out of DNA…….( gene involved in making ……..is isolated / obtained using reverse transcriptase from
mRNA/ synthesise gene using data base )
3.Vector is needed which is the plasmid / virus used / micropipette injection/ gene gun/ liposomes .
4. Insert the gene into vector
5. and DNA ligase link the sugar phosphate back bone allowing incorporation of gene
6. Insert the vector into …….using heat shock/ gene gun/ microinjection .
 GMO
1. Gene
A) cut the gene using restriction enzyme
B) obtain the gene using reverse transcriptase where mRNA act as template to make cDNA
C) synthesizing gene using data base

2. Vector …..plasmid / virus / liposome ……….micropipette injection / gene gun

Insert gene into the vector

3. If vector is plasmid …DNA ligase sugar phosphate back bone

4. Insert the vector into the host cell …using heat shock / gene gun / microinjection

Insert gene
Host cell is bacteria
Steps. For genetically modified crop ( golden rice )

1.identify the gene involved in making beta carotene in bacteria / synthesis the gene using database / obtain nthe
gene using reverse transcriptase
2. Use restriction enzyme
2.Used to cut beta carotene gene out of DNA of bacteria
3.Vector is needed which is the plasmid / virus used / liposomes .
4. Insert the gene into vector
5. Plasmid ( recombinant DNA) / vector put Into rice embryos by heat shock / gene gun / microinjection .
7. Grow rice into adult plants and select the modified rice plants with high yield of beta carotene .
How can u insert a recombinant DNA into other cells

( plasmids , virus, liposomes , microinjections, gene gun ) vectors.

Vectors transfer the required gene , with any marker genes , into new cells

Using virus

- Using Viruses as vectors: viruses bind to receptors on the host cells and insert their genetic material inside the
cell.

Challenges :
1, can cause an immune response in some people as other pathogenic organisms
2. Small packaging capacity / can carry small amount of DNA.
3. Low probability of integration into host genome .
4. Can cause mutation in host DNA / gene insertion disrupts gene function.

B. Using liposomes ( liposome wrapping)

a liposome is a sphere formed of phospholipid bilayer that can fuse with the cell membrane of target cell
and release its contents (recombinant DNA) inside the cell. This is known as Liposome wrapping.

Cause fewer side effect and less potential immune responses ….even
though not very effective at transferring (DNA Challenges : low ability to
add genes into target cells )
Microinjection :

Use a fine glass micropipette to physically inject the desired DNA into a fertilsed egg

Not very efficient because many cells have to be injected before one
accepts the DNA successfully …yet this method has resulted in most
successful transgenic animals .

Gene gun

Shoot a DNA carried on a very small gold pellets into cells at

high speed
Some cells survive this treatment and accept DNA as part of
the genetic material .
Knock out organism

A knockout, as related to genomics, refers to the use of genetic engineering to inactivate or remove one or more
specific genes from an organism. Scientists create knockout organisms to study the impact of removing a gene from
an organism, which often allows them to then learn something about that gene's function

Knock out organism …..is an organism with one or more gene silenced ( knocked out) so no longer works
A) to identify the function of the gene
Genome sequencing identify many genes , but yet scientist dont know the function of the gene in an organism.

B) to investigate disease and test potential treatments . …

Knock out gene coding for non functional protein in human …to creat animal models of the
disease ..these animals can be used for understanding human diseases and ways of treatment .
Where you can test drugs which are unethical for use with humans
Larger sample can be used in testing

Suggest the advantage of using mice as a knock out model compared with human model

Control genetic make since human shows genetic variation

Thus allowing one gene function to be investigated
besides mice reproduce quickly so get quicker results
Gene knock out ….complete elimination of gene from an
Where you can use drugs which are unethical for use with humans organism …no longer works
Larger sample can be used in testing Knock down …reduction of the gene expression
Drugs from genetically modified organisms

A How can u produce insulin from genetically modified bacteria

1. Extract mRNA for insulin from pancreatic beta cells .

2. Then mRNA is incubated with the enzyme reverse transcriptase ( obtained from retroviruses ) .
3. Using mRNA as a template to make cDNA
4. Then cDNA is converted into double stranded DNA molecule using DNA polymerase to assemble nucleotides
5. Where the sticky ends are created using restriction endonuclease and the plasmids are obtained and cut using
same restriction enzyme ( EcoR1) …leaving complementary sticky ends .
6. Then mix the insulin gene with plamsid
7. Some of the plasmid sticky ends pair up with the sticky ends of the insulin gene …using ligase which links
together the sugar phosphate backbone of DNA molecule and the plasmid forming RECOMBINANT DNA

Advantages for producing insulin in this way

1. More effective beacuse its identical to human insulin
2. More rapid response.
3. No iummune response
4. Highly pure so no risk of infection being transferred with insulin

5. Cheaper for larger volumes

6. Has fewer ethical and moral objections because animals are not involved in its production
7. Good for people who had developed tolerance to animal insulin
B Growth hormone ..known somatotrophin
Secreted by pituitary gland
Stimulate growth

C Factor VIII …for blood clotting

Factor VIII is called anti hemophiliac factor .

These people with hemophilia ( poor blood clotting due lack of this factor VIII) ….they need regular injection of VIII

In old time they uses donated blood….carry risks for infections

Advantages :
Over coming all problems that can come from blood donations
A) where extracting the factor from blood is difficult as needs many donations to get just small amount of
factor VIII
B) overcoming the risk that factor VIII could be contaminated with disease from donor .
So recombinant DNA technique produce much more factor VIII, more easily and without risk of disease
contamination.
C) over come any ethical objection that people may have to extract and transfer blood / human material
from one person to another .
D Banana vaccine

Vaccine is a very effective way of elimination of diseases

Problem with poor countries , cant afford vaccines
A) required storage in fridge
B) health care workers needed to vaccinate the children

We can use genetically modified plants or plant products such banana , potatoes and carrots
Where they carry vaccines against human disease such as diarrhea or hepatitis B
Advantages
Local communities could grow these modified plants
Which would be relatively cheap and no need for cool storage
 Use of genetically modified plants

1. Extract the Ti plasmid from Agrobacterium

tumefaciens .
2. Insert the gene of interest into the Ti
plasmid using ligase enzyme and insert back
the plasmid ( recombinant DNA) into Transgenic plants
bacterium by heat shock.

3. Infect the plant with the modified bacterium

So that part of the Ti plasmid with the engineered gene become part of the plant plant chromosome.
4. A. tumefaciens Causes tumor to develop on the plant , where these plant cells contain the new gene .
5. Tumor cells are taken and cultured , where a whole new plant can be grown from them ( produce transgenic
plants ).
 Compare bet ween producing a drug from genetically modified plant with that from
genetically modified microorganism .

Drugs from GM plants Drugs from GM microorganisms

Required gene is isolated Required gene is cut from human

from any living organism or any other organism and
and inserted into Ti plasmid inserted into plasmid
of A. Tumefaciens

Plant cells infected by modified Plasmid is transferred into the bacterial host cells where it
bacteria ..transfer the desired gene becomes part of the bacterial DNA ..
to the whole plant genome
Plasmid ….bacteria
Plasmid…..bacteria ….plant

Plant cells are cloned on a suitable media to produce a Transformed bacteria are identifie'd using gene marker …they are
mass of undifferentiated modified plant cells / plant transferred and cultured in fermentors to make the new protein .
cells are grown on a culture to produce new modified
plant cells containing new gene .
-

Plant cells then transferred to suitable Downstream processing required to

medium to produce huge numbers of GM separate the microorganisms and the
plantlets that will mature to produce the desired end product
desired drug in their leaves/fruit etc.
8/ 4/2023
Microarray
Uses of genetical engineering / GM organisms

A . Human

Production of proteins such as insulin, GH , factor VIII

So they are human proteins that dont stimulate an immune response / dont cause allergies

Many people think that the use of GM bacteria in the production of drugs such as
insulin is a very good thing that does not damage the bacteria, but that brings a lot of with
benefit to many people and makes a number of drugs more accessible to people.
Some people think that changing the genetic material of any organisms is interfering
with nature / the will of their God and feel it is unethical and wrong, even if it does
save many lives and improve the quality of life of many people.
against
B. Plants

1. Flood resistant rice .

Jano
2. Plants that are GM to be pest resistance ( inserting a gene that codes for a toxic
substance that kill pests before infecting plants )
3. Modifying plants that are resistant to herbicides . …example Soya beans
Were we use A. Tumefaciens and gene guns to add new gene to soya beans .
4. Changing and increasing nutrients value of plants .
Example soya beans ( GM plants )
A) herbicide resistant to common weed killers
B) fatty acid balance

Normally it contains little oleic acid and stearic acid , large amount of linoleic acid .
Linoleic acid oxidise easily so no longer good for eating

GM soya beans has lots of oleic acid and less linoleic acid …The changed balance of fatty acids in soya beans
benefits the producers because there is a higher percentage of oleic acid. This does not oxidise easily so the soya
beans and their products last longer before going off. This benefits the producers.

Oleic acid is a monounsaturated fatty acid and there is some evidence that it is better for the health than
linoleic acid. So, using products made with the modified soya beans means consumers have potential health
benefits as well as possible price reductions because the beans have a longer shelf life.

C . Animals

Investigate the use of genetically modified animals to produce human medicines …select one
drug and evaluate the success of this process so far

Blood clotting factors such as factor VII and IX from goat / sheep / rabbit milk
Alpha 1 antitrypsin from sheep ( for protection of lungs )
ATryn ( anti thrombin ) ..used in treating hereditary antithrombin deficiency , from goat milk
Evaluation will depend on drug chosen , it should include assessment of cost of producing the drug, effect on animals
used , success of procedure used to creat a transgenic animals , benefits to people who are treated with the drug
compared with previous treatment

Concerns for using genetically modified plants or animals:

1. Animals might give animals an unfair competitive feature over others so cause disruption of food chain .

2. For humans
Open the door to gene manipulation which is ethically un accepted

3. Plants being resistant to herbicides might cross pollinate with a wild relative resulting in the production of super
weeds which are hard to control

4. Might be abused to be used in the production of biological weapons .

5. Only available in certain countries , this lead to discrimination .
6. Strong objections to use animals in scientific research . …unethical
7. Infertile seeds …..as scienetists add marker genes in GM plants ..these marker genes may include
alleles that ensure the plants is infertile so plant cant reproduce
So remove risk of them spreading in the environment
But also this means that farmers must buy fresh supplies of transgenic plant seeds each year for planting
Disadvantage for poor countries ….

8. Antibiotic resistance being used as a gene marker

1. Risk the spreading resistance to other bacteria …
2,. So antibiotics become no longer effective .

9. Risk of eating alien DNA in GM food plants

GM food contains vital medicines or important vitamins …benefit balance shift dramatically
Also there is growing evidence that eating GM food carries no risk and many benefits so in time it would
be no longer of concern. …
Advantages of using plants that have been Disadvantages of using plants that have been genetically
genetically modified modified
1. Cross pollinate with wild relative
1. Tolerant to climate changes such as drought
2. Producing a new more resistant weeds ( super weeds )
resistant .
3. Loss of traditional variety
2. Plants that are GM to be pest resistance so less
4. Loss of genetic diversity
J-4Monoculture.
pesticide used .
5. unknown long term effect of eating GMO / might
3. Modifying plants that are resistant to herbicides . …
have allergic reactions in human so may be unsafe to
example Soya beans reduce competition from weeds
human
4. Changing and increasing nutrients value of plants .
6. Less food available for other animals so affecting rest
5. Flood resistant rice
of food chain
6. Crops can be grown in poor quality land without the
7. GM seed could be difficult to obtain for farmers in
need of fertiliser….gm crops adapted to unfav conditions.
7. Improve food quality . developing countries
8. Cost effective to farmers 8. high cost of buying GM seed …too expensive for
9. Less effect on food chains and pollinators ..so beneficial to farmers to buy as they may have to buy seeds every
environment. season .
10. Produce GM plants that produce vaccine such as GM
9. May not well in all conditions
banana contain vaccine against diarrhea and hepatitis B
10. Under developed countries becoming more dependent
on other countries .

Accumulation of genetically engineered material in the

environment / other species
 Microarrays

D A technique used …….in two ways :

1. Comparing the gene present in complete genomes of two different species ( gene analysis )
2. Finding out which genes are expressed in tissues or cells at any given time ( gene expression profiling)

Is small piece of glass or plastic that has many thousands of tiny spots in known
positions, each spot has many copies of ssDNA with known sequence of bases
Features
1. Thousands of spots are arranged in rows and columns on solid surface of glass , silicon
or plastic .
2. Microarrays can be the size of a microscope slide , or even smaller ( 2 Cm2)
3. Each spot on a microarray contain multiple copies of a single stranded DNS ssDNA.
4. DNA sequence on each spot is unique and known as probe.
5. Each spot represent a single gene or part of a specific gene .
6. The exact location and sequence of each spot is recorded in a computer database ( data
base holding all details about these DNA probes for many genes )
 Using microarray technique to identify genes that are actively transcribed in the……
. Finding out which genes are expressed in tissues or cells at any given time ( gene expression profiling)

1. The mRNA from two types of cells ( diseased and

undiseased ) is extracted and collected …sample
collection and isolation of mRNA
2. And then use reverse transcriptase to use mRNA as
template to produce cDNA
3. Then cDNA is labelled with fluorescent tags
….denatured to give single stranded DNA ……creation
of labelled cDNA
4. and allowed to hybridise with probe on microarray ……
5. each probe is unique to a different gene
6. Spots on the microarray that fluoresce indicate the
genes that were transcribed in the cell ( indicating genes
expressed ) …… ( 4, 5, 6 …hybridisation)
9. The intensity of light emitted by each spot indicate the 7. Collection and anaylsis using bioinformatics/
level of activity of each gene ….high intensity indicates 8. Compare the sequence of the active genes from large
that many mRNA were present in the sample…..and number of individuals with and without disease to
vice versa ( light intensity is proportional to degree of identify mutation
expression) …..thus knowing activity of gene which is
use of bioinformatics / algorithms to analyse the (enormous qua
needed in treating cancer .
of) data (from the microarray) (1) /
Bioinformatics

It’s the development of the software and computing tools that stores , organise and analyses data about the
genomes (DNA sequences) of living organisms.

- Bioinformatics is aiming for :

Facilitate access to large amount of data
Facilitate access to data on DNA and proteins.
Large data base …..where we Use data base to find probes
Fast , accurate ……Using computer soft wares and Statsticl anaylsis

1. When a genome has been sequenced, comparison can be made with other genome …..Compare whole genomes

:
across species to work out evolutionary relationships…. .
2. Also Similarity and comparison can be made between amino acid sequences of proteins or structures of
proteins …close similarities indicate recent ancestors.

3. Compare whole genomes across individuals in one species to find links between genes and diseases…

4- Predict the primary structure of proteins from gene sequences and visulaise the 3D structure ..to identify the
function…so help find a drug that can bind / block the activity / disrupt the structure of protein .

5. To find out when and where genes are expressed during a development .
Mutations in other genes can result in blindness. Describe how these genes could be identified.

1. Using microarray technique to identify genes that are actively transcribed in the eye .
2. . genome of individuals with inherited forms of blindness is sequenced, …..using bioinformatics
3. comparison can be made with other genome …..compare the sequence of active genes from a
large number of individuals with and without blindness to identify mutation .

Steps
1. The mRNA from cells in eye is extracted and collected ( from large number of
individuals with and without blindness ) …sample collection and isolation of mRNA
2. And then use reverse transcriptase to convert mRNA into cDNA …
3. The cDNA is labelled with fluorescent tags ….and allowed to hybridise with
probe ion microarrays ….creation of labeled cDNA
5. Each probe is unique to a different gene .
6. Spots on the microarray that fluoresce indicate the genes that were transcribed
in the cell ( indicating genes expressed ) .. hybridisation
7. Collection and analysis using bioinformatics
 Answered DR. Nihal Gabr
'
(c) Mutations in other genes can result in blindness.
Describe how these genes could be identified.
(3)

Use microarrays technique , to be able to identify genes that are active /

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

switched on and transcribed in the eye .

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

The activity of many genes can be anaylsed in a single sample .

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

Genome of individual with inherited forms of blindness is sequenced .

I
. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

…..compare the sequence of the active genes from a large number of

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

individuals with and without blindness to identify mutations .

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

Technique
. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

Sequencing( data base )

. . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................................................................................................................................ .............. .. . . . . . . . . . . . . . . . . . . . .

Comparison
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(Total for Question 5 = 10 marks)

Steps
1. The mRNA from cells in eye is extracted and collected ( from large
number of individuals with and without blindness ) …sample collection
and isolation of mRNA
2. And then use reverse transcriptase to produce cDNA using mRNA as
template …
3. The cDNA is labelled with fluorescent tags ….and allowed to hybridise
with probe ion microarrays ….creation of labeled cDNA
5. Each probe is unique to a different gene .
6. Spots on the microarray that fluoresce indicate the genes that were
transcribed in the cell ( indicating genes expressed ) .. hybridisation
7. Collection and analysis using bioinformatics
Genome of the individuals with and without …… is sequenced .
/ Using bioinformatics or data base to analyse the data

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*P67793A02032*