6.3 Flashcards
What is Somatic Cell Gene Therapy?
Somatic Cell Gene Therapy involves inserting functional alleles into body cells.
what is the basic principle of gene therapy
the basic principle of gene therapy is to insert a functional allele of a particular gene into cells that contain only mutated and non-functioning alleles of that gene. If the inserted allele is expressed then the individual will produce a functioning protein and no longer have the symptoms associated with the genetic disorder
what has knowledge gained from the human genome project allowed for
it has led to further possibilities such as using interference RNA to silence genes by blocking translation.
when does Cystic Fibrosis occur ?
cystic fibrosis occurs when an individual inherits two faulty recessive alleles for a particular gene, as a result the differentiated cells where this gene should be lack the protein product of that gene.
if the functioning alleles for this gene are put back into specific cells so that these cells can make the protein then these cells will function normally
Who does Somatic Cell Gene Therapy benefit?
Somatic cell gene therapy affects only certain types of cell types.
The alterations made to the patient’s genome are not passed to the patient’s offspring..
How can Somatic Cell Gene Therapy treat Cystic Fibrosis?
Patients with Cystic Fibrosis lack a functioning CFTR gene.
The alleles can be packaged within a spherical lipid bilayer to make Liposomes.
Liposomes are placed into an aerosol inhaler and sprayed into the noses of patients.
The Liposomes will pass through the plasma membrane of the cells lining the respiratory tract.
If they also pass through the nuclear envelope and insert into the host genome, the host cell will express the CFTR protein - a transmembrane chloride ion channel.
why does cystic fibrosis need to be treated regularly with somatic cell gene therapy
Epithelial cells lining the respiratory tract are replaced every 10-14 days, so this treatment has to be repeated at regular short intervals.
How can Viruses be used in Somatic Cell Gene Therapy?
Viruses have been used as vectors.
If a virus that usually infects humans is genetically modified so that it encases the functioning allele to be inserted into the patient, whilst at the same time being made unable to cause a disease, it can enter the recipient cells, taking the allele with it.
What are the issues with using Viruses as vectors as gene delivery agents?
- Viruses may still promote an immune or inflammatory response
- The patient may become immune to the virus, making subsequent deliveries difficult or impossible
- The Virus may insert the allele into the patient’s genome in a location that disrupts a gene involved in regulating cell division, increasing the risk of cancer
- The Virus may insert the allele into the patient’s genome in a location that disrupts the regulation of the expression of other genes
How can Artificial Chromosome be used in Somatic Cell Gene Therapy? (current research)
Genes can be inserted into an artificial chromosome that would co-exist with the other 46 chromosomes in the target cells.
What is Germ Line Gene Therapy?
Germ Line Gene Therapy involves inserting functional alleles into Gametes or Zygotes
What are Germ Cells?
Reproductive cells that give rise to sperm and ovum
Who does Germ line Gene Therapy benefit?
The cells of the individual will be altered permanently, so their offspring will also inherit the foreign alleles.
What are the issues Germ line Gene Therapy?
There are concerns about how the genes potentially being inserted into a location that could potentially disrupt the expression or regulation of other genes or increase the risk of cancer.
Germ line Gene Therapy is considered ethically impermissible for humans.
What is Genetic Engineering?
Genetic engineering is also known as Recombinant DNA Technology, because it involves combining DNA from different organisms.
It is also called Genetic Modification.
Genes are isolated from one organism and inserted into another organism, using a suitable vector.
What is Recombinant DNA?
Recombinant DNA is a composite DNA molecule created ‘in vitro’ by joining foreign DNA with a vector molecule such as a plasmid.
What are the Four main Stages of Genetic engineering?
- The required Gene is Obtained
- A copy of the gene is placed inside a vector.
- The vector carries the gene into a recipient cell
- The recipient expresses the novel gene
How can we obtain the required gene using mRNA?
mRNA can be obtained from cells where the gene is being expressed.
An enzyme, Reverse Transcriptase can then catalyze the formation of a single stand of complementary DNA (cDNA) using the mRNA as a template.
The addition of primers and DNA polymerase can make this cDNA into a double stranded length of DNA, whose base sequence codes for the original protein.
Where are Reverse Transcriptase Enzymes present in Biological Life?
Retroviruses, such as HIV, use Reverse Transcriptase enzymes that catalyse the production of cDNA (complementary DNA) using their Viral RNA as a template.
How can we obtain the required gene if we know the nucleotide sequence?
- The Gene can be synthesised using an Automated Polynucleotide Synthesiser.
- if the sequence of the gene is known, Design Polymerase Chain Reaction (PCR) primers to amplify the gene from the genomic DNA.
How can we obtain the required gene using a DNA probe?
A DNA probe can be used to locate a gene within the genome and the gene can then be cut out using restriction enzymes.
what are restriction enzymes
endonuclease enzymes that cleave (split) DNA molecules at specific recognition sites
How can we place the gene into a Plasmid vector?
Plasmids are obtained from bacteria and mixed with Restriction Enzymes that will cut the plasmid at specific recognition sites.
The cut plasmid has exposed unpaired nucleotide bases, called sticky ends.
If free nucleotide bases, complementary to the sticky ends of the plasmid, are added to the ends of the gene to be inserted, then the gene and cut plasmid should anneal (bind).
DNA ligase enzyme catalyses the annealing.
A gene mu be sealed into an attenuated (weakened) virus that could carry it into a host cell
Where else are Ligase Enzymes used in Biology?
DNA ligase enzyme is used in molecular biology to join DNA fragments.
It catalyses condensation reactions that join the sugar groups and phosphate groups of the DNA backbone.
These enzymes catalyse such reactions during DNA replication in cells and are also used in the PCR.
Where are Restriction Enzymes present in Biological Life?
Restriction Enzymes are Endonuclease Enzymes that cleave DNA molecules at specific recognition sites.
Bacteria and Archaea have restriction enzymes to protect them from attack by phage viruses.
These enzymes cut up the foreign viral DNA, by a process called restriction, preventing the viruses from making copies of themselves.
The prokaryotic DNA is protected from the action of these endonucleases.
How can we place the gene into a Viral vector?
A gene may be sealed into an attenuated (weakened) virus that could carry it into a host cell.
How can we place the vector in the Recipient Cell?
DNA does not easily cross the recipient cell’s plasma membrane.
Various methods can be used to aid the process:
- Heat shock treatment
- Electroporation
- Electrofusion
- Transfection
- Small pieces of Gold or Tungsten are coated with the DNA and shot into the plant cell using a gene gun
- Plasmids are inserted into certain bacterium which naturally inserts its genome into the host cell genomes.
how does heart shock treatment allow for a vector to be placed into a recipient cell
if bacteria are subjected to periods of hot and cold, and the presence of calcium chloride, their walls and their membranes will become more porous and allow in the recombinant vector. This is because the positive calcium ions surround the negatively charged parts of both the DNA molecules and phospholipids in the cell membrane thus reducing repulsion between the foreign DNA and the host cell membranes
What is Electroporation?
Electroporation involves using high voltage to disrupt the cell membrane, making it more porous and allowing the introduction of a novel gene into a cell.
What is Electrofusion?
Electrofusion involves using electrical fields help to introduce DNA into cells.
What is Transfection?
Transfection involves DNA being packaged into a bacteriophage, which can then transfect the host cell.
How have Scientists obtained insulin from GM Bacteria?
Scientists can obtain mRNA from beta cells of islets of Langerhans in the human pancreas, where insulin is made.
- Adding reverse transcriptase enzyme makes a single strand of DNA and treatment with DNA polymerase makes a double strand - the gene.
- Addition of free unpaired nucleotides at the ends of the DNA produces sticky ends.
- Now, with the help of ligase enzyme, the insulin gene can be inserted into plasmids extracted from E. coli bacteria. These are now called recombinant plasmids, as they contain inserted DNA.
- E. coli bacteria are mixed with recombinant plasmids and subjected to heat shock in the presence of calcium chloride ions, so that they will take up the plasmids.
how do we ensure that transformed (transgenic) bacteria that have resistance to some antibiotics dont escape into the wild
we knock out a gene which means they can not synthesise a particular nutrient
Genetic Manipulation technology:
what are the advantages and disadvantages of modified microorganisms
+GM microorganisms can make human insulin to treat all diabetics
+human growth hormone can treat people with pituitary dwarfism
-could escape into the wild and transfer marker genes for antibiotic resistance
Genetic Manipulation technology:
what are the advantages and disadvantages of modified soya beans
+GM soya beans were made resistant to a herbicide so that competing weeds would die and they wouldnt
-gene for herbicide resistance could pass into weeds
Genetic Manipulation technology:
what are the advantages and disadvantages of modified golden rice
Genetic Manipulation technology:
what are the advantages and disadvantages of modified pathogens
+ genetically modified viruses can be used to make vaccines as they still have antigens but wont make recipient ill
+modified viruses can also act as vectors in gene therapy
-some problems with use of viruses in gene therapy as the allele may be inserted into the genome in a way that increases the risk of cancer or interferes with gene regulation
Genetic Manipulation technology:
what are the advantages and disadvantages of modified pharmaceutical proteins
+human pharmaceutical proteins can be inserted into animals and produced in their milk
What is Electrophoresis?
Electrophoresis is a procedure that separates proteins or DNA fragments according to size or electrical charge.