University of Arkansas System
Type of paper: Thesis/Dissertation Chapter
Transgenesis and Selective Breeding
The relation between humans and genetic manipulation is older than we think. Humans have been manipulating the transfer of genetic information between organisms for over 10.000 years. The first experiences were with cultivation of grains and domestication of animals. The facilities that these methods bring in order to keep having the necessary stuff for our survival make the humans improve their techniques. Now, with the advances of science, we have some sophisticated ways to make easier get the most wanted kinds of livestock and plants. Selective breeding and transgenesis are examples of popular (and successful) processes involving genetic manipulation in the current context.
Nowadays, with the many abilities of the science, techniques are improving livestock. One of them is the ability to engineer and altered DNA from organisms. These organisms are termed Genetically Modified Organisms (GMOs) and may be modified one of 3 ways: by alteration of existing gene, by deletion of existing gene or by addition of foreign genetic material. The last one enables the GMO to express the trait coded by the new gene. These organisms are referred to as transgenic.
The aims of transgenesis can be for specific economic traits or for disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied).
The transgenic cows are an example of transgenic animals. As a transgenic animal, the transgenic cows have the extra gene (transgene) present in every cell, but it’s only expressed in mammary tissue, making the transgenes protein only found and extracted from the cow’s milk. In New Zealand, the AgResearch have been successfully producing transgenic cows that make modified milk or produce therapeutic proteins to treat human diseases.
Making a transgenic cow is a multi step process. Scientists who produce transgenic cows use a range of techniques including DNA cloning, restriction enzymes, ligation, polymerase chain reaction (PCR), transformation, nuclear transfer and in vitro embryo production. In New Zealand, AgResearch have been doing diverse researches about transgenic cows. Now, with these, it is possible to simplify the technique to do transgenic cows in 7 steps:
Step 1: Identification of trait
First, the scientists make an analysis in order to solve problems and find the characteristics wanted in the transgenic animal. After decided the kind of livestock required, it is searched where it’s possible to find the transgene and how to align it logically.
Step 2: Sourcing the transgene
The desired gene sequence is extracted from the source organism’s DNA. The scientists obtain the sequence from a genomic library, that’s a collection of cloned segments of DNA containing at least one copy of every gene from a particular organism. The DNA product contains the organism’s entire DNA sequence, thus it is the desired trait plus the rest of the organism’s DNA.
Step 3: Gene Isolation
Once the gene has been indentified and located, the scientists need to remove the gene sequence from the rest of the DNA. With restriction enzymes, the DNA is cut leaving a bunch of pieces with varying lengths. One of which is the gene of interest. It will be with sticky ends in order to be easily glued back into a vector. This way, the transgene will have the specifically variant that is needed.
Restriction Enzymes come from bacteria and are used as a defence mechanism. When viruses (or other bacteria) attack, bacteria kill them by cutting up in both strands of DNA, at a specific sequence, usually about 4-8 base pairs long.
Step 4: Designing and constructing the gene
After isolated, the transgene is made modifying parts of the gene. The gene construct is a unit of DNA that includes:
A) A selectable marker gene: Usually an antibiotic resistance gene. This is added in order to select cells that have successfully taken up the gene construct.
B) A promoter sequence: A tissue-specific promoter sequence is used to correctly switched the start of expression from the protein in cells with appropriate tissue, for example, mammary cells in lactating cows.
C) The desired gene
D) A terminal sequence: A terminal sequence is needed to signal the cellular machinery that the end of the gene sequence has been reached.
It all is connected with a ligation enzyme and mixed. This product is incubated in the water bath at 16 degrees for half an hour. Then, the scientists use the PCR (Polymerase Chain Reaction).
Polymerase Chain Reaction is a technique that allows scientists to copy and multiply a piece of DNA millions of times. The DNA is heated to 98ºC so that is separates into single strands and polymerase enzyme is added to synthesis new DNA strands from supplied nucleotides.
Step 5: Transformation into bovine cells
The gene construct is incorporated into the genome of a cow cell using a technique called transformation. Transformation involves the delivery of a transgene into the nucleus of a recipient cell and integration into a chromosome so it can be passed onto offspring. Since cows have billions of cells, it would be impossible to insert a copy of the transgene into every cell, so tissue culture techniques must be used.
Tissue culture is the technique of obtaining samples of tissue, growing it outside the body without a scaffold, and reapplying it A bovine cell line is cultured in an incubator. During the transformation, holes are made in the cell membrane allowing the DNA to enter. The holes can be made by applying an electrical pulse or by adding chemicals to the cells. Once inside the cell, the gene construct may enter the nucleus and incorporate into the cell’s genome. That can be done either by using an actual stimulus that interferes with the membrane and allows for a short time for the DNA to enter a cell or just by chemical reactions reagents that again interfere with a membrane that surrounds the cell and then allows temporarily for a DNA molecule to enter.
The recipient genome is exposed to the transgenes in hopes that a few of the transgenes will actually be integrated into that recipient genome and then properly expressed. This is a rarely case and that’s why the next step involves selection of cells expressing the transgene. There is also concern that transformation might indirectly after the expression of other genes because of the unpredictable integration of transgene resulting in a toxic phenotype.
Transform a bovine cell line is necessary because inject the transgene directly into a cow will only change the somatic cells, and the aim is affect the gametes to pass onto the offspring.
Step 6: Selecting for transgene positive cells
To know if the gene has successfully incorporated, it is needed to screen the cells. The cells are transferred to a selective growth medium containing an antibiotic or chemical, depending on which selectable marker was used. After the antibiotic or chemical is added, the cells that haven’t taken up the transgene will die. The other will survive because they contain an antibiotic resistance gene, making them resistant. The survivors will divide and form a small colony of identical cells.
Then, it’s involved Polymerase Chain Reaction (PCR) to photocopier and runs off a whole lot of copies of the gene in order to visualize that the transgene is actually present. The two strands in the DNA double helix need to be separated in a denaturation, done by raising the temperature of the DNA solution. This causes the hydrogen bonds between the complementary DNA chains to break, and the two strands separate. Next, the temperature is lowered and an enzyme joins free DNA nucleotides together. The order in which these nucleotides are joined to the new strand is determined by the sequence of nucleotides in the original DNA strand which is being copied. The result is a double stranded DNA molecule which contains one newly made strand and one original strand. After, the newly created double helix is separated (by heating the solution) and the cycle is repeated.
The cells are also tested by Southern Blotting, which includes DNA digestion, gel electrophoresis technique, blotting, probe labelling, hybridization & washing and detection. To perform it, the bovine cell DNA is digested by restriction enzymes and run out on a gel. The DNA is denatured into single strand DNA and transferred to a piece of nylon membrane. Then a radioactive DNA probe is made containing the DNA sequence of the transgene of interest. The paper is rinsed with the probe, and if the probe is identical to any DNA sequence on the paper it will bind to it. Finally, the paper is exposed to X-ray film. A band or mark on the film indicates that the gene of interest is integrated into the bovine cell DNA.
Step 7: Making a transgenic embryo using nuclear transfer and cloning Nuclear transfer is used to create a whole animal from a single transgenic bovine cell. The generation of a transgenic calf follows the same process as the
generation of a cloned calf. Ovaries are collected from cows processed at the local abattoir. Eggs are removed from the ovaries and matured overnight in a special media. The nuclear material is then removed from the egg using a fine glass needle and a single cultured cell (carrying the transgene) is positioned against the cytoplasm of the egg (injection). The transgenic bovine cell is fused with a bovine oocyte (egg). An electrical pulse is applied to help fuse the cells. The reconstruct (egg + fused cell) is then chemically activated and placed into culture for development to begin. Once fused with the oocyte, the transgenic cell’s chromosomes are reprogrammed to direct development into an embryo. After 7 days, the transgenic embryo will become a blastocysts and will have about 150 cells, so they can be transferred into a recipient cow for further development to term.
If the embryo develops to full term, after 9 months, the cow will give birth to a calf. To confirm that the calf is transgenic, scientists can check using:
1. Polymerase Chain Reaction (PCR) – PCR can quickly establish whether the transgene is present or absent in the calf’s DNA.
2. Quantitative PCR (q-PCR) – q-PCR is to quantify how many copies of the transgene have been incorporated into the genome of the cell line. The q-PCR machine is a standard PCR but with the incorporation of a fluorescent dye that shows the amplification of the DNA product live on screen as the reaction carries out.
3. Fluorescence in-situ hybridisation (FISH) – FISH is a technique in which include take a biopsy from the animal, grow up cells back into culture, arrest them at metaphase and prepare some slides with those cells. With the slides is possible to probe where the transgene is in the chromosome and visualize if it has integrated into more than one chromosome.
4. Analysing of protein expressed – When cows are two years old they may have their first calf, this way it is stimulated the lactation and milk production. At this point, the milk can be tested to determine whether transgenic proteins, like casein and myelin basic protein are present.
Assuming the transgene has successfully integrated itself into the genome, it will be present in every cell of the animal that develops and will be passed on to following generations through regular sexual reproduction.
Interestingly, the creation of transgenic animals has resulted in a good turn of events. Transgenic technology holds great potential in many fields, including agriculture, medicine, and industry. The impact of transgenic animals reaches ecosystems, genetic biodiversity, health and survival of individuals, populations and evolution of populations. Some of the implications of the transgenic process are very important as:
Impact over genetic biodiversity, health or survival of individuals and populations Improving livestock and animal health Transgenic technologies could be used to improve animal health by increasing resistance to diseases. When technology using molecular biology was developed, it became possible to develop traits in animals in a shorter time and with more precision. In addition, scientists can improve the size of livestock genetically. Transgenesis can allow larger herds with specific traits.
Improving food quality or making novel food products
Improving the quantity or quality of the milk or meat from cows may be of value. For example, milk with extra casein requires less processing to make into cheese and will have increased calcium levels.
AgResearch’s first transgenic cows had extra bovine kappa casein genes inserted in their genome. This research proved to the scientists that transgenic technologies could be used to alter milk composition in cows. In the future, modified milk from transgenic cows could be used to benefit animal health, for example, by improving growth and survival of calves, prevent animal diseases, such as mastitis, make milk with human health benefits, assist milk processing into dairy products. Overseas milk or meat products from transgenic animals are not allowed to enter the animal or human food supply in New Zealand.
Creating therapeutic proteins
Transgenic cows can be used as ‘biofactories’ to produce human therapeutic proteins. Therapeutic proteins are used to treat human diseases and they include hormones, antibodies, vaccines, growth factors and blood clotting factors.
In June 2006, the first therapeutic protein made in a transgenic animal was approved for use in Europe and the USA. ATryn®, a human antithrombin protein, is made in transgenic goats. The protein prevents blood clots in patients who don’t make their own version of this protein. Products such as insulin, growth hormone, and blood anti-clotting factors have already been obtained from the milk of transgenic cows too. Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis. The A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.
Scientists at AgResearch have generated transgenic cows that produce myelin basic protein (MBP) in their milk. MBP is part of the insulating layer that surrounds nerves. In patients with multiple sclerosis, this insulating layer is gradually destroyed, which prevents the nerves from communicating. Treatment with human MBP may help reduce symptoms of multiple sclerosis.
Impact over ecosystems
In New Zealand, to start a research as the transgenic cows by AgResearch, it is needed to follow strict guidelines for care and containment of the animals. Transgenic cows are classed as new organisms and are regulated by the Hazardous Substances and New Organisms (HSNO) Act. The HSNO Act is overseen by the EPA, the Environmental Protection Agency. The EPA provides rules and regulations for introducing any hazardous substances or new organisms to New Zealand. Before any research can be done, an application must be made to the Environmental Protection Authority (EPA). EPA evaluates the benefits and risks of any research and decides whether the work can begin. Anyone can make a submission on an application, which can support it, oppose it or support some parts and oppose others. Applications to EPA can be viewed on the EPA website.
ERMA may place restrictions or require certain standards to be followed before giving approval for transgenic research work. For example, the transgenic cows at AgResearch are kept in a special containment facility at Ruakura with restricted access and environmental monitoring. Beyond, transgenic animals cannot leave the facility and the farmers must follow strict rules for waste disposal.
The animal containment facility is monitored by the Ministry of Agriculture and Forestry (MAF) New Zealand. All waste materials from the transgenic cow facility must be disposed of on site. Milk is treated by fermentation, then diluted and sprayed over the pasture. After consultation with local Māori, it was agreed that all animal carcases would be buried on site.
Impact over society
Ethics is a crucial part of the nature of biotechnology. Transgenic animals can contain genes that would not normally arise through natural genetic variation. In New Zealand, transgenic technologies are highly regulated, with all genetically modified animals being kept in containment. However, using or adapting an animal raises issues about animal welfare, the environment, human health and wellbeing, and society. This issue may be viewed differently by different stakeholder groups according to their cultural, spiritual or religious beliefs and values.
As part of the HSNO Act, scientists need to consult with Māori at a local and national level through meetings or hui. Together, they consider the risks and benefits an application may pose to Māori culture or traditional relationships with ancestral lands, water, sites, wāhi tapu, valued flora and fauna or other taonga.
The ethics thinking tool must be used before any decision is made: Consequences – what are the benefits and risks?
Rights and duties – what rights need to be protected and who is responsible for this? Autonomy – should individuals have the right to choose for themselves, or does one decision count for everyone? Virtue – what is the ‘good’ thing to do?
Multiple perspectives – what perspectives do groups with other cultural, spiritual or religious views have?
Ethical concerns must be addressed as the technology grows, including the issue of lab animal welfare. The research must consider all the factors and people involvement to this, never think in the individual but in the society. The future direction of transgenic research will be influenced by ongoing discussion and evaluation of ethical and societal issues that are raised. New Zealanders need to weigh up the risks and benefits associated with transgenic cows and decide what they consider to be acceptable.
Selective breeding of animals is a selective mating to increase the possibility of obtaining certain characteristics in the animals in order to get better livestock. The type of mating selected depends on the goals. To produce the kinds of animal they want, breeders have to first understand the animal as a species, then the animal as genetic individuals. Selective breeding use many techniques as outcrossing, linebreeding, inbreeding and hybrids. The more modern techniques involve a wide variety of laboratory methods, including embryo selection, artificial insemination, cloning and MOET.
1. Outcrossing – Mating two animals that are unrelated for at least 4 to
6 generations back is called an outcross. This method works best when the genetic variation for a trait is high.
2. Linebreeding – Linebreeding involves mating related animals like half-brother/half-sister, cousins, aunt/nephew, and other more distant relationships.
3. Inbreeding – This breeding method involved mating directly related animals, like mother/son, father/daughter, and full brother/full sister (full siblings). This method is used generally to create uniformity and prepotency (the ability of this process to continue) and to force out latent weaknesses from the gene pool.
4. Hybrid – First generation cross between two animals that belong to different breeds. Hybrid is process that occurs in nature, particularly in plants. However, humans have learned how to manipulate the genes in a similar way using the same principles. With increased rate of mutations, offspring are selected that contain the genetic variation that suites the desired need. Hybrids contain a unique number of chromosomes when compared to distant relatives of similar genomes. The hybrids then carry traits of both species.
5. Composite – Two hybrids of same breed-combination bred back to each other for generations.
1. Embryo Selection – Embryo Selection is used to select the best embryos according the livestock wanted. Embryo Selection is crucial in horticulture and agriculture.
Sex Selection: Sometimes, one gender tends to be preferred for a specific purpose. Sex selection is vital for the production of offspring. a. Females are useful in commercial purposes eg) ju, dairy cows b. Males that are able to breed with many females to pass on desired traits; expensive if the cows are inseminated.
2. Embryo Manipulation – Embryo Manipulation takes place not long after fertilisation and beginning of the zygote process of mitosis (morula stage). The new cells formed are called blastomeres and they are totipotent from the 4 to 8 cell stage. In this time, scientist can manipulate the embryo in order to get some desired characteristics.
3. Artificial Insemination – Artificial insemination is the artificial introduction of semen from a male with desirable traits into females of the species to produce pregnancy, and is useful because a far larger number of offspring can be produced than would be possible if the animals were traditionally bred.
4. Multiple Ovulation and Embryo Transfer (MOET) – MOET is the production of multiple embryos from a female with desirable traits, which are then implanted in the wombs of other females of the same species.
5. Cloning – Cloning, an asexual method of reproduction, produces an individual with the same genetic material (DNA) as another individual. Animals have been cloned by three processes: embryo splitting, blastomere dispersal, and nuclear transfer. Nuclear transfer is most common and involves enucleating an ovum, or egg, with all the genetic material removed.
Selective breeding programmes have resulted in higher yields and better disease resistance. Ultimately, breeding goals are dictated by market demand; however, it is not easy to predict what consumers will want several years in advance. Although it is extremely effective, there are disadvantages to this method. One of these is that for animal breeding to be performed productively, a number of animals must be involved in the process. Another problem is that undesirable traits can also mistakenly be selected for. For this reason, too much inbreeding will produce sickly or unproductive stock, and at times it is useful to breed two entirely different strains with each other. The resulting offspring are usually extremely healthy; this is referred to as “hybrid vigor.” Usually hybrid vigor is only expressed for a generation or two, but crossbreeding is still a very effective means to combat some of the disadvantages of inbreeding. Another practical disadvantage to selective inbreeding is that the DNA of the parents is altered during the production of eggs and sperm. In order to make eggs and sperm, which are called gametes, a special kind of cell division occurs called meiosis, in which cells divide so that each one has half the normal number of chromosomes (in humans, each sperm and egg contains 23 chromosomes). Before this division occurs, the two pairs of chromosomes wrap around each other, and a phenomenon known as crossing over takes place in which sections of one chromosome will be exchanged with sections of the other chromosome so that new combinations are generated. The problem with crossing over is that some unexpected results can occur. For instance, the offspring of a bull homozygous for two recessive but desirable traits and a cow with “normal” genes will all have one copy of each recessive gene. But when these offspring produce gametes, one recessive gene may migrate to a different chromosome, so that the two traits no longer appear in one gamete. Since most genes work in complicity with others to produce a certain trait, this can make the process of animal breeding very slow, and it requires many generations before the desired traits are obtained—if ever.
The evolution of scientific methods has been contributed lot through time. In the agricultural and horticultural environment, the transgenic and selective breeding methods have been improved livestock and better animal/plant health. Beyond, the researchers can contribute in fields such as medic and industrial.
The techniques in both processes stimulate knowledge and improve the technology, resulting in employment and better conditions to the future. However, transgenesis and selective breading involve the manipulation of the natural order, bringing a polemic topic.
But, if the scientists follow the ethic process and rules determinate by superior agencies, always thinking about the society over the individual, they may have good results, supporting the future generations. Therefore, all the opinions about the topic must be discussed and respected.
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