Genetic Engineering PDF

Genetic Engineering Some techniques use to propagate organisms with desirable traits are as follows: Traditional Technique: Artificial selection (selective breeding, inbreeding, hybridization) Modern Technique Reproductive Cloning Recombinant DNA technology (use in Gene Cloning)  Artificial...

Genetic Engineering Some techniques use to propagate organisms with desirable traits are as follows: Traditional Technique: Artificial selection (selective breeding, inbreeding, hybridization) Modern Technique Reproductive Cloning Recombinant DNA technology (use in Gene Cloning)  Artificial Selection ❖Modification of species by human intervention in order to produce organisms with desirable traits in succeeding generations (i. e. plant and animal breeding) ❖A biological process by which humans select preferred visible traits in organisms and breed them to produced offspring with desired traits. ❖Humans select organisms with certain phenotypic trait values for breeding. They cannot control what genes are passed. When they get offspring with the desired traits, they maintain them. Selective Breeding ❖ when organisms with desired characteristics are mated to produce offspring with desired traits. ❖Passing of important genes to next generation. ❖Examples: ❖champion race horses, cows with tender meat and produced more milk, large juicy oranges ❖Dachshund were once bred to hunt badgers and other burrowing animals.  Examples of selective breeding: Angus cows are bred to increase muscle mass so that we get more meat, Egg-Laying Hen-produces more eggs than the average hen Inbreeding ❖breeding of organism that are genetically similar to maintain desired traits. ❖Dogs breeds are kept pure this way. ❖Its how a Doberman remains a Doberman. ❖Risk: since both have the same genes, the chance that a baby will get a recessive genetic disorder is high. ❖Risks: blindness, joint deformities. Hybridization ❖two individuals with unlike characteristics are crossed to produce the best in both organisms. ❖It may involve crosses between different species (interspecific hybridization) or crosses between genetically different individuals (breeding lines or cultivars) or within a species (intraspecific hybridization). ❖Example: Luther Burbank created a disease resistant potato called the Burbank potato. He crossed a disease resistant plant with one that had a large food producing capacity. Examples of hybridization: 1. Liger: lion and tiger mix 2. Grape + apple= grapple. The fruit tastes like grapes and looks like apple. Cloning ❖Reproductive and Therapeutic cloning use Somatic Cell Nuclear Transfer (SCNT) ❖A mature somatic cell is fused to an enucleated egg cell “in vitro”. The egg cell which contains the somatic cell’s nucleus is stimulated using electric shock in order to divide and grows into an embryo. This is then implanted into the uterus of a foster mother and developed into organism with the same characteristics like that of the donor of somatic cell. ❖Remember one diploid cell has all the DNA needed to make an entire organism. ❖Each cell in the body has the same DNA, but cells vary because different genes are turned on in each cell. Cloning ❖In general, cloning is a technique to create exact genetic replica of genes, cells or individual organism ❖Gene cloning- creates copies of genes or segment of DNA segment ❖Reproductive Cloning- creates copies of whole animals ❖Therapeutic Cloning- creates embryonic stem cells ❖Reproductive cloning is creating an organism that is an exact genetic copy of another. ❖identical twins are naturally created clones. ❖Clone: group of cells or organisms that are genetically identical as a result of asexual reproduction ❖They will have the same exact DNA as the parent. ❖Dolly, a sheep, was the first mammal that was successfully cloned. Dolly was born 5 July 1996 to three mothers (one provided the egg, another the DNA and a third carried the cloned embryo to term) Dolly: The Sheep Benefits of cloning: 1. You can make exact copies of organisms with strong traits. 2. Increases food supply 3. Medical purposes: clone organs for Saber Tooth Tiger (extinct) transplants 4. It may prevent species from getting extinct. Disadvantages of cloning: 1. Decreases genetic diversity 2. If one of your clones gets a disease, they all get it: same immune system. 3. Inefficient: high failure rate: 90%+ 4. Expensive  Genetic Engineering ❖is a process that uses laboratory-based technology to alter the DNA of an organism ❖This may involve changing, deleting or adding a new segment of DNA ❖Genetically Modified Organism (GMO) or transgenic organism is genetically engineered to produce new traits that are desirable. ❖Examples: bacteria that produce human insulin, Flavr Savr tomato, HB4 soybean, Bt rice and Bt corn ❖Bt stands for Bacillus thuringiensis, a gram positive, soil dwelling bacterium. It is used as insecticide against different insect pests in agriculture. Ernst Berliner isolated a bacteria that killed Mediterranean flour moth in 1911. He named it as Bacillus thuringiensis after a German town Thuringia where the moth was found. Bt crops are resistant to pests. Bt proteins (toxins) target specific insects. Gene Cloning ❖A target gene is cut out (gene splicing) from the DNA of one organism and inserted to a DNA of another organism using restriction enzyme (molecular “scissor”). ❖For example: human insulin gene can be inserted in bacterial plasmid so that bacteria can make human insulin. Gene Cloning 1. A restriction enzyme cuts the desired gene (i.e. gene that codes for insulin) from DNA of an organism (i.e. human DNA) 2. A plasmid is removed from a bacterium and is cut with the same restriction enzyme. (A plasmid is used as vector to introduced the desired gene to a bacterium (Plasmid is a circular DNA in bacteria) How are genes cut for gene splicing? ❖A restriction enzyme cuts the DNA at a specific code. ❖There are thousands of restriction enzymes. Each one cuts DNA at a different sequence. Some look for GGCC and cut in between the G and C. Every time GGCC is found in the DNA it is cut by the restriction enzyme. TTATGGCCATACGGCCTT AATACCGGTATGCCGGAA. TTATGG CCATACGG CCTT AATACC GGTATGCC GGAA ❖This DNA segment was cut twice creating three fragments. ❖Since every one is different, we all have different amount of times GGCC is found. Gene Cloning Plasmid with insulin gene 3. The desired gene is inserted into the bacterial plasmid (vector) 4. The “recombinant” plasmid is placed back into the bacteria. 5. The cell now has directions (DNA) to make insulin. Thus, when bacterium multiplies, it can produced more insulin. (Its human insulin, bacteria do not make insulin on their own, thus they are called GMOs or transgenic organism since their genetic make up has been modified or altered) ❖Cloned gene may be amplified ”in vitro” using PCR (Polymerase Chain Reaction) to create many copies of such genes. Benefits: insulin is cheaper There are no side effects because it is human insulin. Pig insulin was once used but there are side effects and the process is more expensive.  BASIC STEPS OF GENETIC MODIFICATION 1. Isolating a gene to be inserted 2. Inserting the gene in a vector (agent used to carry foreign gene) 3. Inserting vector into the host 4. Multiplication of host cells by cloning 5. Extraction of desired product Transgenic Animals ❖ Genes are inserted into these animals so they will produce what humans need. ❖A way to improve the food supply ❖i.e. Transgenic cows genes are inserted to cow to increase milk production Spider goat ❖gene from spider is inserted into goat ❖Goats make silk of the spider web in their milk. ❖Flexible, stronger than steel; used in bullet proof jackets. Glow-in-the-dark cats Scientist used a virus to insert DNA from jellyfish The gene made the cat produce a fluorescent protein in its fur. Bioluminescent frog It’s a miracle of genetic engineering. You can see through the skin; how organs grow; how cancer starts and develops without dissecting the frog. The GloFish was the first genetically modified animal to become available as a pet. It is a natural Zebrafish which has genetic information from bioluminescent jellyfish added to its DNA. Zorse is a cross between a zebra and a domestic horse. The crosses were originally done in England and Africa in an attempt to produce a domestic horse like animal that was resistant to diseases spread by a fly in Africa. Transgenic bacteria: gene inserted into bacteria so they produce things humans need. For example: insulin and clotting factors in blood are now made by bacteria. Transgenic Plants Transgenic Plants ❖ Plants are given genes so they meet human needs. ❖i.e. Transgenic corn : given a gene so corn produces a natural pesticide. Now they don’t have to be sprayed with cancer causing pesticides. 25% of all corn is like this. Venomous cabbage ❖Gene from a scorpion tails is inserted into cabbage ❖Cabbage now produces that chemical. ❖It limits pesticide use while still preventing insects from damaging crops ❖Corporations say that the toxin is modified so it isn’t harmful to humans. Banana vaccines ❖virus is injected into a banana, the virus DNA becomes part of the plant. ❖As the plant grows, it produces the virus proteins — but not the disease part of the virus. ❖When people eat a bite, their immune systems create antibodies to fight the disease — just like a traditional vaccine ❖Vaccines for hepatitis and cholera The Flavr Savr tomato was a tomato engineered to have a longer shelf life. In 1995, Bt Potato was approved safe by the Environmental Protection Agency.  Bt-Cotton is a genetically modified cotton which is resistant to pests. Golden Rice genetically modified to contain beta-carotene (a source of Vitamin A). Transgenic fruit obtained from pear and apple. A Blue Rose is a genetically modified Rose. In Medicine: Vaccination generally involves injecting weak live, killed or inactivated forms of viruses or their toxins into the person being immunized. Gene therapy is the genetic engineering of humans by replacing defective human genes with functional copies.  In Agriculture. ❖ Improved nutritional quality. ❖ Better nitrogen fixation. ❖ Disease resistant plant. ❖ Enhanced efficiency of minerals used by plants to prevent early exhaustion of soil fertility. ❖ Reduced post harvest losses.  In bioremediation. ❖ Bioremediation can also be accelerated by creating a super-bug that contains genes from different types of petroleum hydrocarbon-degrading bacteria so that one microbe species can fully break down spilled petroleum. ❖ The first genetically modified organism patented in the US was a petroleum-metabolizing super-bug developed by an Indian scientist named Ananda Chakrabarty in 1980. ❖ At the time, Pseudomonas was the only genus of bacteria known to break down pollutants. ❖ To make a Pseudomonas super-bug, Dr. Chakrabarty combined the DNA of four different species of petroleum- degrading Pseudomonas to generate a new species called Pseudomonas putida.  Genetic engineering has the risk of introducing allergens & toxins into safe foods. Transgenic technology is also being used to remove the allergens from peanuts, one of most serious causes of food allergy. The Food and Drug Administration (FDA) checks to ensure that the levels of naturally occurring allergens in foods made from transgenic organisms have not significantly increased above the natural range found in conventional foods. ❖A virus is often used to deliver DNA. ❖In the movie “I Am Legend,” a healthy gene was inserted into a virus. ❖The virus invaded the cancer cells and inserts the healthy gene to cure cancer. ❖Worked at first but the virus mutated and became deadly. ❖This is being attempted in real life.  Gene therapy ❖A technique that uses a gene(s) to treat, prevent or cure a disease or medical disorder. ❖ It works by adding new copies of a gene that is broken, or by replacing a defective or missing gene in a patient's cells with a healthy version of that gene. ❖A medical approach that treats or prevents disease by correcting the underlying genetic problem. ❖ It allows doctors to treat a disorder by altering a person’s genetic makeup instead of using drugs or surgery. The earliest method of gene therapy, often called gene transfer or gene addition, was developed to: Introduce a new gene into cells to help fight a disease. Introduce a non-faulty copy of a gene to stand in for the altered copy causing disease.  Gene therapy ❖A newer technique, called genome editing (an example of which is CRISPR-Cas9 short for Clustered Regularly interspaced short palindromic repeats and CRISPR- associated protein 9), uses a different approach to correct genetic differences. Instead of introducing new genetic material into cells, genome editing introduces molecular tools to change the existing DNA in the cell. ❖Genome editing is being studied to: Fix a genetic alteration underlying a disorder, so the gene can function properly. Turn on a gene to help fight a disease. Turn off a gene that is functioning improperly. Remove a piece of DNA that is impairing gene function and causing disease. Gel Electrophoresis ❖A laboratory technique used in molecular biology to separate and identify molecules such as DNA, RNA and proteins. ❖DNA samples are loaded into wells at one end of a gel and an electric current is applied to pull them through the gel. ❖DNA fragments are negatively charged thus they move towards the positive electrode. Fragments are separated according to their size. Small fragments move faster than large ones. ❖When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each represents a group of same sized DNA fragments. ❖The resulting bands may be used to compare DNA of two or more individuals. DNA sample is loaded into the wells of a machine filled with gel. The gel is spongy and the DNA squeezes through the pores. The machine is plugged in and the fragments get separated based on their size. The smaller fragments move farther than the large fragments.  Separation of DNA based on size of fragments. Final result of gel electrophoresis Why compare DNA? 1. To solve paternal disputes 2. In forensics, to solve criminal cases 3. To determine how closely species are related (to provide evidence of evolutionary origin)  Related Video Links Genetic Engineering https://www.youtube.com/watch?v=jAhjPd4uNFY DNA Fingerprinting https://www.youtube.com/watch?v=V7FM5FJ1vxc Gene Cloning https://www.youtube.com/watch?v=rYkegqhXLbE Gene therapy https://www.youtube.com/watch?v=4YKFw2KZA5o Genome Editing https://www.youtube.com/watch?v=trjPZKmxMJI CRISPR https://www.youtube.com/watch?v=MnYppmstxIs https://www.youtube.com/watch?v=TdBAHexVYzc https://www.youtube.com/watch?v=KSrSIErIxMQ https://www.youtube.com/watch?v=IiPL5HgPehs