Biotechnology

Definition

 Use of living systems and organisms to develop/make useful products: Any

technological application that uses biological systems, living organisms, or

derivatives thereof to make/modify products or processes for specific use

Cloning
 Cloning - the DNA of one organism is copied exactly into another organism

o Genes are copied within the same species

Types of cloning

 Embryo cloning/embryo splitting - splitting cells apart from a developing

embryo before they become specialized to produce several embryos.

 Identical twins are natural clones.

 Nuclear transfer - transferring a body cell into an egg and then stimulating

cell division to produce an embryo

 Cuttings - taking a small piece of stem or leaf and growing it in the right

conditions to produce a new plant

o Cut a branch off and plant it in damp compost

o Plant hormones are often used to encourage new roots to develop

o Cutting is covered in a clear plastic bag to keep it moist and warm

 Tissue cloning - getting a few cells from a desirable plant to make a big mass

of identical cells, each of which can produce a tiny identical plant

o Grown in vitro (in glass tubes/Petri dishes) using sterile agar jelly that

contains plant hormones and nutrients

o More expensive than cuttings

o Used to preserve rare plant species or grow commercially larger nurseries

 Clones are also naturally produced via asexual reproduction

Dolly the Sheep

 1996
 The cell nucleus was removed from an egg cell from a Scottish Blackface

ewe

 Adult cells from the udder of a 6-year-old Finn Dorset were cultured and

injected into the egg cell

 A spark of electricity fused the udder cell with the egg cytoplasm and

stimulated the egg to grow into an embryo in the womb of a surrogate sheep

Pros of cloning
 Producing new babies for infertile couples

 Organ transplants won’t be rejected

 Ensure the survival of endangered species and bring back extinct animals

 Producing useful proteins in milk to treat diseases

 Producing medically useful animals

 Producing prize agricultural animals

 Pet cloning

Cons of cloning

 Many embryos miscarried before Dolly

 High embryo wastage

 Religious/ethical beliefs that it is wrong to interfere with nature

 Wrong to destroy/tamper with embryos

 Reduce genetic variation

Selective Breeding

 Selective breeding - modifying gene pools by selecting favourable

characteristics, providing us with improved crops and livestock

o Ex: disease resistance or improved milk yield

 Humans have created hundreds of varieties of different species, generally

used for food, by selecting desirable characteristics in organisms and then

 cross-breeding them with similar individuals.

o Desirable characteristics: disease-resistant food crops, wheat plants that

produce a lot of grain, large or unusual flowers

o New varieties may be economically beneficial, provide more or better quality

food, and allow farmers to feed more people.

 Takes many generations

The steps of selective breeding

 Decide which characteristics to select

 Choose parents that show these characteristics and breed together

 Choose the best offspring with desired characteristics to produce the next

generation

 Repeat the process over many generations

 Desirable traits: animals that produce a lot of milk or meat, chickens that lay

large eggs, domestic dogs that have a gentle nature

Pros

 Economically beneficial and higher quality food

 Animals can be bred to cause no harm, ex: cattle without horns

Cons

 Reduced genetic variation → attack of disease/insect → extreme destruction

 Rare disease genes may be selected as a part of a desired trait → problems

with specific organisms

 May create physical problems in specific organisms, ex: overbred dogs

 Genetic Engineering
 Genetic engineering/genetic modification - DNA is modified so a unique set

of genes is produced

o Genes can be swapped across species.

 Direct manipulation/modification of DNA so that a unique set of genes is

produce

 A faster way to modify species by directly transplanting genes for a desired

trait into an organism

 We can genetically modify many organisms, such as bacteria or cows.

 A major part of Genetic engineering includes moving a piece of DNA from

one genome to another.

 To change an organism's DNA, the desirable gene must be located, isolated,

copied, and introduced to the new genome.

 The big difference between genetic engineering and selective breeding is

that with genetic engineering, scientists can introduce genes from a

completely different species into an organism's genome.

o These organisms are called transgenics

Recombinant DNA Technology

Tool kit

 Restriction enzyme - found in bacteria and used to destroy viruses; cut DNA

at a particular sequence, leaving “sticky ends”

o Sticky ends - when a staggered cut is made in DNA by a restriction enzyme,

the exposed unpaired bases can pair with complementary bases on another

strand of DNA which has been cut with the same restriction enzyme

o Exposed ends = sticky ends

 DNA ligase - connects pieces of DNA together; can be used to “stick” a gene

into a vector such as a plasmid

 Plasmid - independently replicating circle of DNA containing a few genes

o Found in bacteria and can be used to carry genes into a suitable host

bacterium

 Vector - something which can carry DNA from one organism to another

 Host bacterium - a bacterium which can be modified by adding genes using a

vector

o Allowed to multiply to produce multiple copies

Steps

 Restriction enzymes isolate the required gene, leaving it with sticky ends.

Sticky ends are short sections of unpaired bases.

 A vector, usually a bacterial plasmid or a virus, is cut by the same restriction

enzyme, leaving it with corresponding sticky ends.

 The ligase enzyme joins together the vector and the isolated gene

 The vector inserts the gene into the required cells

 The genes are transferred to animal, plant or microorganism cells during

early development, which allows them to develop with the desired

characteristics.

o Reproduction occurs in an incubated fermenter.

Benefits

 Agriculture - see GMOs

 Create insulin which can be used to treat diabetes

 Sterile insects, such as mosquitoes, could be created. They would breed with

fertile mosquitos but be unable to reproduce. This would reduce the number

of offspring and may help with the spread of diseases, such as malaria,

dengue fever and the Zika virus.

Risks

 A gene that benefits one organism may harm another

 Not ethical
 Genetically engineered crop seeds are usually more expensive, so people in

developing countries can’t afford them.

Genetically Modified Foods

 GMO - genetically modified organisms

 In some countries, vitamin deficiencies are common

o Insert genes that add nutrients into plants such as rice so that they have

vitamin A, iron, etc…

 Golden rice - gene produced beta carotene (needed in humans to make

vitamin A)

o Can help reduce the occurrence of deficiency diseases

 Include those that are resistant to insect attack or are herbicide resistant →

increased yields

o Herbicide-resistant crops can tolerate the herbicide, but the weeds can’t, so

they’re killed

 Ethics

o Health risks

 It may contain substances which cause allergic reactions in some people

 Higher levels of toxins may be found in the food

 Long-term effects are not known

o Some think it is ethically wrong to create new life forms or to move genes

between different species

o Pollen produced by the plants could be toxic and harm insects that transfer it

between plants
o However, it may help reduce hunger around the world by increasing crop

yields

 It’s also faster than selective breeding

 Transgenic plants

o Crops are added with herbicide/pesticide-resistant gene

o 98% of soybeans in Argentina are genetically modified

Stem Cell Research

 Stem cells are the cells of the early embryo from which all the cells in the

adult body will develop (ESCs)

o They are totipotent (Can differentiate into any specific cell type)

o Other types of stem cells in body: bone marrow/certain blood cells (ASCs)

o Used to replace worn out or faulty cells

o These are pluripotent and can only differentiate into only one type of

specialized cell

 Bone marrow transplants - have been in use for over 20 years

o Patient’s bone marrow cells are replaced with those from a health, matching

donor

o If successful, cells migrate into the patient’s bone marrow and produce nex,

healthy blood cells to replace the old faulty cells

 Applications

o Replacing damaged tissue

o Screening toxins
o Development of treatment that allows people paralyzed because of spinal

injuries to walk again

o Possibility of growing replacement organs that are matched to the patient

o Genetic disorders to be studied and new drugs to treat them can be tested

 EMCs are highly controversial

o People think it is unethical to use embryonic stem cells since they can turn

into baby

 They think it’s “meddling with nature.”

o Others say its unethical to ignore EMCs since research can help save lives -

remove paralysis, regrow an amputated limbs, etc

 Heart disease: Adult bone marrow stem cells can be injected into heart

arteries and may improve cardiac function in victims of heart failure/heart

attack

 Type I diabetes: pancreatic cells do not produce insulin, so stem cells may be

able to reproduce healthy pancreatic cells which can secrete insulin

Organ Printing
 Process where artificial organ can be created using 3d printer/bio printer by

utilizing stem cells

 Use ink jet needles to deliver cells

o Sticky polymer to form support structure

o Pressurized air

 Small electric field is applied that converts the mixture into superfine threads

with cells trapped inside

 Method usually utilizes the layer-by-layer method to create tissue like

structures that are later used in medical and tissue engineering fields

Challenges

 Selection of correct cells

 Materials to project these onto

 Use of factors that help stem cells grow and differentiate in the correct way

 Technical challenges related to sensitivities of living cells

 Proper construction of tissue

Steps

 Stem cells from the patient are put in a growth medium so that they can

multiply

o When enough cells are available, they are put into bioprinter cartridges

 Stem cells form “bioink” which, once loaded into cartridges, can be used in

the bioprinting process

o Cartridge is made from a chamber attached to a long extrusion nozzle fitted

with a syringe

 Bioprinter is driven by software that lays down the stem cells in precise

patterns

o Cells can be put down in layers, interspersed with a water-based material

called hydrogel, which forms a temporary mould around the cells

o Sticky polymers can also be used to scaffold the cells

 Scaffolding material is laid down by separate nozzles on the bioprinter

 These sticky polymers are printed through fine inkjet nozzles

 Printed tissue is left to grow

 Printed tissue can be used in transplants or in medical research

 View Hodder pg. 293 for diagram

 Lab-grown tissues and organs can be made by creating artificial scaffolds in

the shape of the desired organ and then by adding living cells to the scaffold

 Problem in creating tissue: vascular system - needed to provide cells with

oxygen and nutrients

Recent situations

 First patent related to this technology was filed in '03 and granted in '06

 '06- world's first artificial bladder was built and transplantation done on 7

patients

 '09- artificial liver was made

 '11 world's first artificial trachea

 '11 In situ skin bioprinting for burn wound was done

 '15 partnership between organovo and l'oreal for commercial production of

artificial skin by using bioprinting technology

Why don’t the organs work?

 Difficult to create blood vessels between tissue layers

 Organs have many specialized functions difficult to replicate

Advantages

 Artificial organ personalized using patient's own cells

 No DNA rejection
 Eliminate need for immunosuppressant drugs after a regular organ

transplant

 Eliminate organ donation

 No waiting period

Disadvantages

 Printers cost hundreds of thousands of dollars

 Possibly more expensive than regular organ transplant

 Use of stem cells is still controversial

 Cost of using stem cells

 No success yet

Genome Mapping
 Genome - the entire genetic material of an organism

o Each diploid cell has one copy of your entire genome

 Finding exact location of specific gene on a chromosome

Human Genome Project: mapped out entire sequence of

human dna

 Began in 1990 and completed in 2003

 13 year effort coordinated by

o Department of Energy (DOE)

o National Institute of Health (NIH)

 Human genome consists of 24 chromosomes and contains over 3 billion

nucleotide pairs

 Average gene consists of 3000 bases

 Sizes of genes vary greatly, with largest known human gene encoding

dystrophic containing 2.5 million base pairs

 3% of genome encodes amino acid sequences and the rest of the genome is

junk

 Functions are unknown for over 50% of the discovered genes

o Opened up ways which genetic disease can be identified

o Ex- CF is on chromosome 7

Importance

 Enables us to search for genes linked to different types of disease

 Opened up new ways in which genetic diseases could be identified and

treated

 Understand inherited disorders and their treatment

 Trace human migration patterns from the past

 Ex: Two examples of these are genes that can contribute to breast cancer,

which are known as BRCA1 and BRCA2

o Mutations in these genes account for approximately 10% of all inherited

breast cancer cases detected

o Scientists detected BRCA1 and BRCA2 genes by studying families where

breast cancer was known to have been inherited between individuals

o They were able to create a pedigree analysis that showed the close

relationship of those affected and unaffected within the family

 Illustrates the inheritance pattern of the disease to be determined

o This enabled scientists to test DNA from the affected and unaffected

individuals to identify differences

o Now possible to detect the presence of the genes by having a simple blood

test

DNA Fingerprinting

 Developed by Alec jeffreys in 1984

 Method that identifies and individual based on patterns formed from

variations in genetic coded

 Also known as dna profiling, genetic fingerprinting, dna typing

 Minisatellites are short sequences (10-60 base pairs long) of repetitive DNA

that show greater variation from one person to the next than other parts of

the genome. This variation is exhibited in the number of repeated units or

‘stutters’ in the minisatellite sequence.

o Humans share about 99.9% of DNA

o Remaining percentage contains around 3 million base pairs

 These differences can be compared and used to help distinguish you from

someone else

Procedure

 Extract DNA from a sample of human material, usually blood

 Restriction enzymes used to cut the DNA → thousands of pieces of DNA of

varying lengths

 Separated according to size by gel electrophoresis

o DNA was loaded into wells at one end of a porous gel, which acted a bit like a

sieve

o Electric current was applied which pulled the negatively-charged DNA

through the gel

 Shorter pieces move easier and faster; longer pieces travel slower

o Electric current is switched off → DNA pieces are separated according to size

 Smallest DNA molecules were farthest away from where the original sample

was loaded onto the gel

 DNA were transferred or ‘blotted’ out of the fragile gel on to a robust piece of

nylon membrane and then ‘unzipped’ to produce single strands of DNA

 Nylon membrane was incubated with radioactive probes

o Probes are small fragments of minisatellite DNA tagged with radioactive

phosphorus

o Probes only attach to the pieces of DNA that they are complementary to – in

this case they attach to the minisatellites in the genome

 Minisatellites that the probes have attached to were then visualised by

exposing the nylon membrane to X-ray film

o When exposed to radioactivity a pattern of more than 30 dark bands

appeared on the film where the labelled DNA was

o This was the DNA fingerprint

 To compare two or more different DNA fingerprints the different DNA

samples were run side-by-side on the same electrophoresis gel

 Uses

 Way of telling individuals of the same species apart

 DNA sequences are variable and can be used as identifying characteristics

 DNA fingerprinting has advantages over other sources of evidence such as

fingerprints, blood type, etc as it is much more accurate

 Good for paternity testing: In the example, first male is father since the

offspring shares half the genes with him

 Criminal cases: eliminating suspects

 Identifying corpse

 Food testing (champagne, horse meat, etc)

 Genetics matching for organ or bone marrow donation