Biotechnology and gene technologies (Pt 3) Bio

• Extracting a specific gene from a donor organism and placing that gene in another organism (often of a different species).
• The organism receiving the gene (recipient) expresses the new gene product through the process of protein synthesis.
• Such organisms are described as transgenic (or genetically modified).

A section of DNA, often in the form of a plasmid, which is formed by joining DNA sections from two different sources.

• 1. The required gene is obtained.
• 2. A copy of the gene is placed (packaged and stabilised) in a vector.
• 3. The vector carries the gene to recipient cell.
• 4. The recipient expresses the gene through protein synthesis.

• mRNA produced from transcription of the gene can be obtained from cells. Eg. mRNA for insulin obtained from Beta-cells Islets of Langerhans in pancreas. mRNA can be used as template to make copy of the gene.
• Gene can be synthesised using automated polynucleotide sequencer.
• DNA probe can be used to locate gene on DNA fragments and gene can be cut from DNA fragment using restriction enzymes.

• Virus genomes
• Liposomes 
• Yeast cell chromosomes
• [CHECK if there are any others!]

• Electroporation - high voltage pulse applied to disrupt membrane so that vector can enter.
• Microinjection - DNA injected using very fine micropipette 
• Viral transfer - uses virus' mechanism for infecting cells by inserting DNA directly.
• Libosomes - DNA wrapped in lipid molecules - so they can cross the lipid membrane by diffusion.
• Heat shock and calcium salts

• A particular restriction enzyme will cut DNA only where a specific base sequence occurs - the sequence is called restriction site. (usually less than 10 bases long, and are palindromic - so base pairs that read the same in opposite directions).
• The enzyme catalyses a hydrolysis reaction, which breaks the phosphate-sugar backbones of the DNA double-helix.
• Gives a 'staggered cut' which leaves some exposed unpaired bases on each end of fragment - a sticky end.

• Vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment containing desired gene, so the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene, so they can anneal.
• Vector DNA and DNA fragment mixed together with enzyme DNA ligase, which catalyses a condensation reaction which joins the phosphate-sugar backbones of the DNA double helix together. (same as in natural DNA replication here).
• Where DNA fragments from different organisms are joined in this way, the resulting DNA ligase is called recombinant DNA.

• engineering
• sequence
• separation

• Improving a feature of the recipient organism. Eg. make plants resistant to herbicides so farmers can use them.
• Engineering organisms that can synthesise useful products. Eg. insulin production

transgenic

Many cut plasmids will, in the presence of ligase enzyme, simply reseal to reform the original plasmid.

• Large quantities of the recombinant plasmid are mixed with bacterial cells.
• Addition of calcium salts and 'heat shock' (where temp of culture is lowerd to freezing, then quickly raised to 40^oC) increase the rate at which plasmids are taken up by bacterial cells.
• However, still very few bacterial cells take up plasmid. Those that do are called transformed bacteria. Bacteria are now transgenic.

• Some colonies that have bacteria that did not take up a plasmid
• Some bacteria that have taken up a plasmid that has not sealed in a copy of the gene, but has sealed up on itself to reform the original plasmid.
• Some - the ones we want - that have taken up the recombinant plasmid - transformed bacteria.

• Orignal plasmids chosen because they carry genes that make them resistant to 2 antibiotic chemicals (ampicillin and tetracycline). These resistance genes are called genetic markers. The host bacteria (E.coli) are susceptible to both of these antibiotics.
• Plasmids cut by restriction enzyme that has its restriction site in the middle of the tetracycline resistance gene, so that if the required gene is taken up, then the gene for tetracycline resistance is broken up and does not work. However, the gene for ampicillin resistance still works.
• Use replica plating:
• Some cells from colonies transferred onto agar that has been made with ampicillin, so only those that have taken up plasmid can grow.
• Some cells from these colonies transferred to agar made with tetracycline so only those which taken up plasmid that does not have insulin gene will grow.
• So now we know that the bacteria which survived in the agar with ampicillin but died in the agar with tetracycline are the ones with the insulin. Now we can take these colonies and grow them on large scale.
• [Another way is inserting fluorescence gene in plasmid along with the desired DNA].

• 1. Transcription in pancreatic cells give mRNA that codes for production of insulin polypeptide.
• 2. mRNA isolated and treated with enzyme reverse transcriptase which synthesises complementary DNA strand - so the template strand. 
• 3. Add DNA polymerase and a supply of DNA nucleotides to single strands so other strand is built and a copy of double-stranded original gene is produced - called a cDNA gene.
• 4. Unpaired nucleotides added to each end to give sticky ends complementary to those on the plasmid after cutting.
• 5. Plasmid then cut open with restriction enzyme and mixed with the cDNA genes. Some of the plasmids take up the gene. DNA ligase then seals up plasmids which are now called recombinant plasmids.
• 6. The plasmids then mixed with bacteria, some of which takes up recombinant plasmids.
• 7. Bacteria then grown on agar plate, and each produce clones, called colony.
• 8. Identify the bacterial colonies that contain the recombinant plasmid by using the marker method described in the last (or so) flashcards. These transformed/transgenic bacteria are cloned in a colony and they eventually produce human insulin.

• Conjugation
• Plasmids often carry genes associated with resistance to antibiotics (as well as those that help bacteria invade hosts).
• Therefore conjugation is advantageous to bacteria because it contributes to genetic variation (esp in case of antibiotic resistance genes), and increase change of survival of the bacteria.

The principles of genetically engineering insulin gene into bacterial cells, which cna then be cultured to produce the polypeptide hormone for human use, can be applied to any similar product. You could be asked to suggest how a supply of any protein might be obtained using genetic engineering and the same broad answer would be appropriate.

• Vitamin A deficiency can often resolt in someone going irreversibly bilnd. Could also affect bones and cell development and epithelial tissue.
• People in less developed countries suffer because of lack of vitamin A.

• beta-carotene
• gut
• beta-carotene
• endosperm
• fat

• 1. 2 genes - phytoene synthase (from daffodil) and Crt1 (from soil bacteria) are needed for the metabolic pathway to make beta-carotene, so are isolated using restriction enzymes.
• 2. Plasmid removed from bacteria and cut open with same restriction enzymes.
• 3. The two genes and a marker gene inserted into plasmid.
• 4. The recombinant plasmid put back into bacteria.
• 5. Rice cells are incubated with the transformed bacteria, which infect the rice cells and insert genes into the plant cell's DNA, creating transformed rice cells.
• The two genes were inserted near to a specific promoter sequence that switches on the genes associated with endosperm development. This meant they were expressed as endosperm grew.
• [NB: Black bit from revision guide, textbook seems to be different but insufficient. ASK TEACHER!!]

• Use of gene technology where the functioning allele of a particular gene is placed in the cells of an individual lacking functioning alleles of that particular gene, so to treat some genetic disorders.
• The transcription of the added working copy of the gene will mean that the individual may no longer have the symptoms associated with the genetic disorder.
• (Can only be used to treat some recessive conditions but not dominant ones like Huntington's).

Body cells that aren't germ cells (ie. sex cells like sperm of ovum), or stem cells.

• Adding genes (augmentation): Engineering functional copy of gene into relevant specialised cells means that the polypeptide is synthesised and the cells can function normally.
• Killing specific cells: Genetic techniques used to make cancerous cells express genes to produce proteins (such as cell surface antigens) that make the cells vulnerable to attack by immune system could lead to targeted cancer treatments.

• Engineering a gene into sperm, egg or zygote or into all the cells of an early embryo means that as the organism grows, every cell contains a copy of the engineered gene. This gene can then function within any cell where that gene is required.
• It can also be passed on to offspring.

• S: Functioning allele of gene introduced into target cells - therefore techniques to get gene to the target location needed, or specific cells must be removed then treated. G: Functioning allele of gene is introduced into germline cells - delivery techniques more straightforward.
• S: Treatment is short-lived and must be repeated regularly because specialised cells containing the gene will not divide to pass on the allele. G: All cells derived from these germline cells will contain a copy of functioning allele, may also be to offspring.
• S: Less ethical issues. G: More ethical issues, as it has permanent effect.
• S: Genetic manipulations restricted to actual patient. G: Genetic manipulations could be passed on to patient's children.

• Genetically modified virus - but problematic because cells become immune to it.
• Liposomes - small spheres of lipid bilayer containing functioning allele. Pass through lipid bilayer of cells and act as vectors to carry allele into cell.

• Virus can bring about immune response. BE CAREFUL! Don't say they are 'rejected'. USE 'rejected' for organs or organisms.
• Virus can affect more than the cells intended.
• High mutation rate.

• Recessive disease
• Disease that is affected by one gene (the smaller amounts of genes the better).

• Transplantation of cells, tissues or organs between animals of different species.
• Allotransplantation means transplantation between animals of the same species?

There is a world-wide shortage of donor organs.

• Genes for human cell-surface proteins are inserted into the animal's DNA. (Injected into newly fertilised animal embryo). Genes integrate into animal's DNA and reduces risk of transplant rejection, as they produce these proteins.
• Genes for animal cell-surface proteins are 'knocked out' - removed or inactivated. These genes are removed/inactivated in nucleus of animal cell, then nucleus is transferred to egg cell which is then stimulated to divide into an embryo so animal created doesn't produce animal cell-surface proteins.

• Differences in organ size
• Lifespan of most pigs 15yrs, so xenograft may age prematurely
• Body temp of pigs is 39^oC (2^oC higher than humans) 
• Possible disease transfer between animals and humans.
• Ethical issues: religious beliefs against use of pigs (eg. Jews)
• Some animal welfare groups strongly oppose killing animals for human use.

• Production of insulin from genetically engineered bacteria
• Golden Rice
• Resistance to pesticides and pests increase yield and help poverty etc.
• Xenotransplantation - help many people who are at risk of dying waiting for donor organ.
• Gene therapy to treat genetic disorders.

• Animal welfare issues that arise from animal suffering during gene manipulations. 
• Religious views on use of some animals (Jews)
• Concerns about germline cell gene therapy: 
• Effects of gene transfer unpredictable
• Germline therapy could also be used to enhance favourable characteristics, leading to concerns about 'designer children' and creation of genetic underclass.
• Others:
• Use of antibiotic resistance genes as markers means these could be passed to other microorganisms, leading to more widespread antibiotic resistance.
• Resistance genes in crop may pass to weeds, forming 'super-weeds'.
• Genes for resistance could have wide-ranging effects on the biodiversity and food chains of the area.
• [Genetic manipulation is a young technology. We lack long-term knowledge of manipulations carried out.]