2.2 Daisy The Cow

RNA interference, or RNAi, is a biological process in which RNA molecules reduce the gene expression of an organism. This is done typically by causing the destruction of specific mRNA molecules. RNAs are direct products of genes, these small RNAs can bind to other mRNA molecules to either increase or decrease their activity like in the example of preventing an mRNA from producing a protein. There are two types of RNA molecules that are central to RNAi, these molecules are, micro RNA (miRNA) and small interfering RNA (siRNA).

In the case of AquAdvantage, they have done this with the Chinook Salmon gene and another gene from the Ocean Pout (the gene from the Ocean Pout has antifreeze proteins. They have inserted this into the gene sequence to allow the Atlantic salmon to grow during the winter months when wild Atlantic salmon stop growing). Restriction enzymes are enzymes which cut the gene from a specific recognition site and are used to extract the desired gene out of the DNA molecule. This gives the DNA “sticky ends” which will allow the sequence to later attach into a circular DNA plasmid. The growth hormone gene is then fused with a promoter such as a metallothionein gene, which allows the growth hormone gene to be produced throughout the body. The DNA fragment then goes through a process called ligation, which is where they are reassembled into a DNA plasmid. The sections of DNA with sticky ends are then joined together by base pairing in a process called annealing. If the ends were blunt, they would not properly attach and the engineered gene could fall apart, and they must have matching base pairs to connect too due to the base pairing rule that only certain bases join with certain bases. They

BSE, Bovine Spongiform Encephalopathy, otherwise known as mad cow disease refers to the changes seen in brain tissue of affected cows. This disease is normally found in adult cows and it slowly but aggressively affects their central nervous system. The U.S. Department of Agriculture (USDA) continuingly does testing on US cattle to prevent this disease from spreading to human consumers. There are several ways the disease can affect the human body, there are ways we can treat it, but most importantly there are ways we can prevent it.

After 40 weeks, the cows were killed and scientist analyzed their tissue. “The organs and glands of the injected animals (thyroid, liver, heart, kidneys, ovaries, and so on) were much larger than in the control group, whereas the total weight of these cows when slaughtered was significantly lower. For example, the right ovary was on average 44 percent heavier in the group having received 5 times the dose than in the control group. The treated cows had significant reproductive problems: whereas 93 percent of the control group were successfully inseminated during the period, only 52 percent of cows from the injected groups were. From one injected animal to another, the hormone level in the blood varied considerably, the fourth group being a thousand times higher than that recorded in the control group.” This document was hidden from the public until it was leaked by an employee who disagreed with the companies evasiveness (Robin 33-34). Though many organic companies label whether there is antibiotic use or GH use in their products, many companies still do not tell the consumers if there is or not.

[see Appendix B.]and also the offal [see Appendix B] of dead cows that carry the

Based on the phenotypic effects observed with either mutant alleles (forward genetic screen) or knockdown effects (e.g. RNAi or morpholino), you determine that you would like to generate a conditional knockout animal (in this case, in a mouse model).

RNA interference also known as gene knockdown was a process used to genetically modify Daisy the cow. RNA interference works by preventing messenger RNA’s from being converted into proteins. The scientist who genetically modified daisy designed micro RNA’s that bind both cow and sheep messenger RNA’s on BLG and tested which micro RNA prevented BLG production. The micro RNA’s were then used to create RNA interference and block messenger RNA’s from being converted into the protein BLG. RNA interference works by double stranded RNA being cut into smaller pieces by an enzyme called ‘dicer’. These small pieces of RNA then bind to proteins called Argonaut proteins which is part of a group of proteins called the RNA induced silencing complex. After this, one strand of the RNA is removed, leaving the other strand to join up with the messenger RNA. Once bound together, the Argonaut protein can then destroy the messenger RNA resulting in a knock down of a certain gene or protein in daisy’s case.

When scientists know what gene they want to manipulate they 'introduce double stranded DNA, or dsRNA to the cell.' The dsRNA produce small double-stranded interfering RNAs, or siRNA into the cytoplasm of the cell.5 from this the expression of the gene decreases drastically but does not get entirely eliminated, therefore showing the role of the targeted gene.

Genetic engineering is the process of manipulating genes by adding new DNA or modifying the genome (which is a made of DNA) to add new beneficial traits in that organism using biotechnology. It removes the DNA from one organism and transfers into another organism. This changes the actions of the genes to serve human goals. DNA plays a huge role in this process. It contains human’s unique genetic code and also hold the instruction to make the proteins in the body.

Genetic engineering techniques have been applied to various industries, with some success. Medicines such as insulin and human growth hormone are now produced in bacteria, experimental mice such as the oncomouse and the knockout mouse are being used for research purposes and insect resistant and/or herbicide tolerant crops have been commercialized. Plants that contain drugs and vaccines, animals with beneficial proteins in their milk and stress tolerant crops are currently being developed

Daisy the cow was born with a birth defect. She had no tail. The process of the genetic transfer is most likely to be the cause of this. Stefan Wagner, a scientist on the team, said "This congenital abnormality is rare in cows and not something we have seen in animals we have cloned previously," However, they are still in the process of confirming whether or not this defect is link to the genetic transferring method. This deformity makes us question whether she has any other health issues. If she was born without a tail, there is a chance she could have other deformities as well. This is not good news as it any form of health problems could affect other parts of the cow.

Cows produce a protein in their milk called BLG and many infants are allergic to this protein. Two to three hundred infants are allergic to cow’s milk, and BLG allergies are seemingly a large part of this. Various organizations are trying to investigate ways to manipulate the genetic material in the modified dairy cattle with the main goal of producing milk that doesn’t contain the lethal BLG protein. Daisy is a genetically modified cloned cow and whom does not produce milk containing the BLG protein but does however have the gene for it. But the mRNA for the BLG protein has become nonfunctional. Once the protein has been manipulated the cow containing the modified gene is cloned to produce a calf with the same genetic material. This cloned cow will then not produce the protein. This technique is very useful to produce a herd without the protein to gather more milk. Because of this protein produced (BLG) this causes a lot of people and infants to become very sick and ill. Therefore by creating a herd of cows using the processes of Gene Knockdown and Cloning this would not only cut out the lethal protein of BLG, but this will also create cows that don’t possess the BLG protein and therefore creating healthy milk for all to drink.

    Through recombinant DNA techniques, scientists have already experimented with bacteria and copied their cellular system that allows them to defend themselves from viruses in order to create a new restriction enzyme called Cas9, which then gets bound to an RNA guide strand. This new enzyme introduces new genetic material into the human genome as DNA replicates. This method will be useful for families members who carry harmful recessive alleles. Their babies could inherit the genetic disorder, but with recombinant DNA techniques, the harmful gene or genes could easily be replaced with a functional gene. Lauren Friedman, Senior editor for

process behind it. This section will explain it a bit better. Genetic modification has been done for

RNAI is the biological process where the RNA molecules inhibit the gene molecule. It works by silencing a sequence of DNA, which is a cellular mechanism. In order for this to happen introduction to the DNA templates to produce short RNAs that are then processed to siRNA. Once they are lode the RNA is silenced, resulting to the loss of the messenger RNA, the end result you can lose a gene expression of a targeted gene.(benitec,2017)