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Transgenic plants with multiple genes?

Transgenic plants with multiple genes?


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I have in mind, as a hypothetical project, to generate a transgenic plant capable of synthesizing vitamin B12. This could be consumed by vegans, for instance, as an alternative to B12 supplements or provide B12 to those that are deficient of it, since this vitamin can only be aquired by consuming animal-based products.

However, after researching a bit, I found out that this vitamin needs at least 30 enzymes for its complete biosynthesis in bacteria. At least 5 of them are already present in plants (the first steps of B12 to siroheme, which is a cofactor also synthesized in plants). The rest of the enzymes (around 20) are only present in bacteria.

Is it possible to make a plasmid containing 20 different gene constructs and transfect it via Agrobacterium transformation? Although I know that plasmids have limited space, so maybe I could use different plasmids, each one with different selection markers?

Thanks in advance.


Upgrade for CRISPR/Cas: Researchers knock out multiple genes in plants at once

In their work, the researchers used markers to distinguish between different plant seeds. No difference can be seen with the naked eye. Under UV light, however, transgenic seeds appear red, non-transgenic seeds green. (left picture) Credit: Jessica Lee Erickson

Using an improved version of the gene editing tool CRISPR/Cas9, researchers knocked out up to twelve genes in plants in a single blow. Until now, this had only been possible for single or small groups of genes. The approach was developed by researchers at Martin Luther University Halle-Wittenberg (MLU) and the Leibniz Institute of Plant Biochemistry (IPB). The method makes it easier to investigate the interaction of various genes. The study appeared in The Plant Journal.

The inheritance of traits in plants is rarely as simple and straightforward as Gregor Mendel described. The monk, whose experiments in the 19th century on trait inheritance in peas laid the foundation of genetics, in fact got lucky. "In the traits that Mendel studied, the rule that only one gene determines a specific trait, for example the color of the peas, happened to apply," says plant geneticist Dr. Johannes Stuttmann from the Institute of Biology at MLU. According to the researcher, things are often much more complicated. Frequently there are different genes that, through their interaction with one another, result in certain traits or they are partly redundant, in other words they result in the same trait. In this case, when only one of these genes is switched off, the effects are not visible in the plants.

The scientists at MLU and IPB have now developed a way to study this complex phenomenon in a more targeted way by improving CRISPR/Cas9. These gene editing tools can be used to cut the DNA of organisms at specific sites. The team built on the work of biologist Dr. Sylvestre Marillonnet who developed an optimized building block for the CRISPR/Cas9 system at the IPB. "This building block helps to produce significantly more Cas9 enzyme in the plants, which acts as a scissor for the genetic material," explains Stuttmann. The researchers added up to 24 different guide RNAs which guide the scissor enzyme to the desired locations in the genetic material. Experiments on thale cress (Arabidopsis thaliana) and the wild tobacco plant Nicotiana benthamiana proved that the approach works. Up to eight genes could be switched off simultaneously in the tobacco plants while, in the thale cress, up to twelve genes could be switched off in some cases. According to Stuttmann, this is major progress: "As far as I know, our group has been the first to successfully address so many target genes at once. This may make it possible to overcome the redundancy of genes," says the biologist.

Until now, creating multiple mutations was a much more complex process. The plants had to be bred in stages with a single mutation each and then crossed with one another. "This is not only time-consuming, it's also not possible in every case," says Stuttmann. The new approach developed at the MLU and the IPB overcomes these disadvantages and could prove to be a more efficient method of research. In future, it will also be possible to test random combinations of several genes in order to identify redundancies. Only in the case of conspicuous changes in the plant's traits would it then be necessary to specifically analyze the genetic material of the new plants.


Free Response

What is the purpose and benefit of the polymerase chain reaction?

The polymerase chain reaction is used to quickly produce many copies of a specific segment of DNA when only one or a very few copies are originally present. The benefit of PCR is that there are many instances in which we would like to know something about a sample of DNA when only very small amounts are available. PCR allows us to increase the number of DNA molecules so that other tests, such as sequencing, can be performed with it.

10.2: Biotechnology in Medicine and Agriculture


Some Achievements

Improved Nutritional Quality

Milled rice is the staple food for a large fraction of the world's human population. Milling rice removes the husk and any beta-carotene it contained. Beta-carotene is a precursor to vitamin A, so it is not surprising that vitamin A deficiency is widespread, especially in the countries of Southeast Asia. The synthesis of beta-carotene requires a number of enzyme-catalyzed steps. In January 2000, a group of European researchers reported that they had succeeded in incorporating three transgenes into rice that enabled the plants to manufacture beta-carotene in their endosperm.

Insect Resistance

Bacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests. Its lethal effect is mediated by a protein toxin it produces. Through recombinant DNA methods, the toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.

Disease Resistance

Genes that provide resistance against plant viruses have been successfully introduced into such crop plants as tobacco, tomatoes, and potatoes.

Figure 16.3.7.1 Tomatoes

Tomato plants infected with tobacco mosaic virus (which attacks tomato plants as well as tobacco). The plants in the back row carry an introduced gene conferring resistance to the virus. The resistant plants produced three times as much fruit as the sensitive plants (front row) and the same as control plants.

Herbicide Resistance

Questions have been raised about the safety - both to humans and to the environment - of some of the broad-leaved weed killers like 2,4-D. Alternatives are available, but they may damage the crop as well as the weeds growing in it. However, genes for resistance to some of the newer herbicides have been introduced into some crop plants and enable them to thrive even when exposed to the weed killer.

Figure 16.3.7.2 Effect of bromoxynil on tobacco courtesy of Calgene, Davis, CA

Effect of the herbicide bromoxynil on tobacco plants transformed with a bacterial gene whose product breaks down bromoxynil (top row) and control plants (bottom row). "Spray blank" plants were treated with the same spray mixture as the others except the bromoxynil was left out.

Salt Tolerance

A large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to grow most important crops. However, researchers at the University of California Davis campus have created transgenic tomatoes that grow well in saline soils. The transgene was a highly-expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf cells. There was no sodium buildup in the fruit.

"Terminator" Genes

This term is used (by opponents of the practice) for transgenes introduced into crop plants to make them produce sterile seeds (and thus force the farmer to buy fresh seeds for the following season rather than saving seeds from the current crop). The process involves introducing three transgenes into the plant:

  • A gene encoding a toxin which is lethal to developing seeds but not to mature seeds or the plant. This gene is normally inactive because of a stretch of DNA inserted between it and its promoter.
  • A gene encoding a recombinase &mdash an enzyme that can remove the spacer in the toxin gene thus allowing to be expressed.
  • A repressor gene whose protein product binds to the promoter of the recombinase thus keeping it inactive.

How they work

When the seeds are soaked (before their sale) in a solution of tetracycline

  • Synthesis of the repressor is blocked.
  • The recombinase gene becomes active.
  • The spacer is removed from the toxin gene and it can now be turned on.

Because the toxin does not harm the growing plant - only its developing seeds - the crop can be grown normally except that its seeds are sterile.

The use of terminator genes has created much controversy:

  • Farmers - especially those in developing countries - want to be able to save some seed from their crop to plant the next season.
  • Seed companies want to be able to keep selling seed.

Transgenes Encoding Antisense RNA

These are discussed in a separate page. Link to it

Biopharmaceuticals

The genes for proteins to be javascript:void('Remove anchor')used in human (and animal) medicine can be inserted into plants and expressed by them.

  • Glycoproteins can be made (bacteria like E. coli cannot do this).
  • Virtually unlimited amounts can be grown in the field rather than in expensive fermentation tanks.
  • It avoids the danger from using mammalian cells and tissue culture medium that might be contaminated with infectious agents.
  • Purification is often easier.

Corn is the most popular plant for these purposes, but tobacco, tomatoes, potatoes, rice and carrot cells grown in tissue culture are also being used. Some of the proteins that have been produced by transgenic crop plants:

  • human growth hormone with the gene inserted into the chloroplast DNA of tobacco plants
  • humanized antibodies against such infectious agents as
    • HIV
    • respiratory syncytial virus (RSV)
    • sperm (a possible contraceptive)
    • herpes simplex virus, HSV, the cause of "cold sores"
    • Ebola virus, the cause of the often-fatal Ebola hemorrhagic fever
    • An example: patient-specific antilymphoma (a cancer) vaccines. B-cell lymphomas are clones of malignant B cells expressing on their surface a unique antibody molecule. Making tobacco plants transgenic for the RNA of the variable (unique) regions of this antibody enables them to produce the corresponding protein. This can then be incorporated into a vaccine in the hopes (early trials look promising) of boosting the patient's immune system - especially the cell-mediated branch - to combat the cancer.

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    Transgenic plants Advantages & Disadvantages

    Transgenic plants are defined as, ” The plants that have been genetically engineered, a breeding approach that uses recombinant DNA techniques to create plants with new characteristics. They are identified as a class of genetically modified organism (GMO)”

    Everyone would love to read about transgenic plants because they are the plants that are created by humankind. But it has to be in a simple definition which should be understood by everyone.

    Transgenic Plants:

    There are many definitions that are told many people. The few definitions of the transgenic plants are:

    The simple definition of transgenic plants is defined as, ” The plants into which one or more genes from another species have been introduced into the genome, using genetic engineering processes.”

    Transgenic plants are plants that have had their genomes modified through genetic engineering techniques either by the addition of a foreign gene or removal of a certain detrimental gene. A foreign gene will be inserted into a plant of a different species or kingdom.

    Transgenic plants are also called Genetically modified crops. It is also defined as, “Genetically modified plants have been engineered for scientific research, to create new colours in plants, deliver vaccines, and to create enhanced crops.

    The transgenic plants are created to fulfil the aim of introducing a new species to the world which doesn’t occur naturally through pollination. The inserted gene sequence is known as Transgene. Plants containing transgenes are often called genetically modi­fied or GM crops.

    Plant genomes can be engineered by physical methods or by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors”.

    The transgenic plants and their products are currently more productive and regulated in the USA under the authority of the U.S. Department of Agriculture (USDA).

    Antibodies are first expressed in Transgenic plants in the year 1989. The various antibodies and antibody fragments and domains have been produced in the plant hosts as well as full length.

    The researchers took an experiment and important research on the development of transgenic plants that are created by micro-organisms or animals which provides the researcher’s insight on what is possible.

    The main reason for creating Transgenic plants is to develop a crop and make it useful and productive as possible.

    It takes time and long process to create a plant with the best genes that are available, or with the closely related species to bring different Genes together.

    The first transgenic plant was created and developed through the insertion of an antibiotic resistance gene into tobacco. After creating the first transgenic plant, it has become popular and many plants are created by the transgene.

    The main purpose of creating transgenic plants is to produce crops, high quality and high yield.

    Crop plants are incorporated with disease resistance gene to confer resistance toward these pathogenic diseases that are caused by pest, bacteria, and viruses.

    In future, the transgenic plants or Generally modified crops will have a valuable alternative in solving the problem of food security that happens in the world of the growing population.

    The unintended effects of gene transfer in GM crops should be examined thoroughly through metabolic profiling methods to avoid the production of GM plant with the significant difference in chemical composition from a non-GM plant or transgenic plant grown under the same condition

    Steps involved in the production of Transgenic plants:

    The few steps that are involved in creating the production of transgenic plants. They are:

    1. Identifying, Isolation and Cloning of Genes for Agriculturally Important Traits
    2. Designing Gene Construct for In­sertion
    3. Transforming Target Plants with the Gene Construct
    4. Selection of The Transgenic Plant Tissue/Cells
    5. Regeneration of the Transgenic Plants

    These are the steps that are involved in the creation of the production of Transgenic plants. The Transgenic plants are created using these steps.

    Transgenic plants Advantages:

    The advantages and benefits of Transgenic plants or Generally modified crops are:

    The main advantages of a transgenic plant or Generally modified crops include larger yield, resistance to diseases and pests and capable of growing under stressful conditions.

    The improvement in yield is one of the advantages of Transgenic plant. The improvement in yield plays an important role in Gene technology and it increased the productivity of food, fibre, crops and vegetable crops.

    The increase in the yield is achieved by controlling losses caused by various insects and diseases.

    Improvement in disease and Insect resistance played an important role in gene technology. The crop plants are infected by insects and the pesticides for the insects. But the transgenic plants or Generally modified crops cannot be affected by the insects.

    Improvement in quality is one of the main advantages of transgenic plants. Gene technology has helped in improving all these three types of quality in different crops. It can produce more in a small area of land.

    It can feed a rapidly increasing population because it shows dramatically increased yields. It reduces the use of pesticide and insecticide during farming that might be great moves for the betterment of the food supply.

    These are the advantages and benefits of Transgenic plants or Generally modified crops which will make you understand about its advantages.

    Disadvantages of Transgenic plants:

    The disadvantages and drawbacks of Transgenic plants or Generally modified crops are:

    The main disadvantages of Transgenic plants are included allergic reactions, the emergence of super-pests and loss of biodiversity.

    It increases the cost of cultivation and more inclined towards marketization of farming that works on immoral profits.

    The transgenic crops endanger not only farmers but also the trade, and the environment as well. It is biologically altered. Hence, biotech foods may pose a human health risk.

    The excessive production of genetically modified foods will be rendered ineffective over time because the pests that these toxins used to deter might eventually develop resistance towards them.

    These are disadvantages and drawbacks of Transgenic plants or Generally modified crops. This will help you know that everything in the world will have their separate pros and cons.

    This article will make you understand to know about the Transgenic plants or Generally modified crops.


    Acknowledgments

    Special thanks to all members of the Center for Agricultural Synthetic Biology at the University of Tennessee for their support as well as laboratory members Lezlee Dice, Taylor Frazier-Douglas, Cassie Halvorsen, Stacee Harbison, Mitra Mazarei, Reginald Millwood, Mary-Anne Nguyen, Alex Pfoetenhaur, Christiano Piasecki, Rebekah Rogers, Yuanhua Shao, Shamira Sultana and Yongil Yang. We sincerely appreciate the assistance from Richard Sexton and Vilmos Magda at the University of Tennessee Pendergrass library with the 3D printing of the custom plant stand. This research was developed with funding from the Defense Advanced Research Projects Agency (DARPA) Award No. HR0011-18-2-0049 and Department of Energy (DOE) Grant No. DE-SC0018347. The views, opinions and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. (Approved for Public Release, Distribution Unlimited).


    Controversies

    The introduction of transgenic plants into agriculture has been vigorously opposed by some. There are a number of issues that worry the opponents. One of them is the potential risk of transgenes in commercial crops endangering native or nontarget species.

    • A gene for herbicide resistance in, e.g. maize (corn), escaping into a weed species could make control of the weed far more difficult.
    • The gene for Bt toxin expressed in pollen might endanger pollinators like honeybees.

    To date, field studies on Bt cotton and maize show that the numbers of some nontarget insects are reduced somewhat but not as much as in fields treated with insecticides.

    Another worry is the inadvertent mixing of transgenic crops with nontransgenic food crops. Although this has occurred periodically, there is absolutely no evidence of a threat to human health.


    Contents

    While German dermatologist Alfred Blaschko described Blaschko's lines in 1901, the genetic science took until the 1930s to approach a vocabulary for the phenomenon. The term genetic chimera has been used at least since the 1944 article of Belgovskii. [3]

    An animal chimera is a single organism that is composed of two or more different populations of genetically distinct cells that originated from different zygotes involved in sexual reproduction. If the different cells have emerged from the same zygote, the organism is called a mosaic. Innate chimeras are formed from at least four parent cells (two fertilised eggs or early embryos fused together). Each population of cells keeps its own character and the resulting organism is a mixture of tissues. Cases of human chimerism have been documented. [1]

    This condition is either innate or it is synthetic, acquired for example through the infusion of allogeneic hematopoietic cells during transplantation or transfusion. [ citation needed ]

    In nonidentical twins, innate chimerism occurs by means of blood-vessel anastomoses. The likelihood of offspring being a chimera is increased if it is created via in vitro fertilisation. [4] Chimeras can often breed, but the fertility and type of offspring depends on which cell line gave rise to the ovaries or testes varying degrees of intersex differences may result if one set of cells is genetically female and another genetically male. [ citation needed ]

    Tetragametic chimerism Edit

    Tetragametic chimerism is a form of congenital chimerism. This condition occurs through the fertilization of two separate ova by two sperm, followed by aggregation of the two at the blastocyst or zygote stages. This results in the development of an organism with intermingled cell lines. Put another way, the chimera is formed from the merging of two nonidentical twins (a similar merging presumably occurs with identical twins, but as their genotypes are not significantly distinct, the resulting individual would not be considered a chimera). As such, they can be male, female, or have mixed intersex characteristics. [5] [6] [7] [8] [9] [4] [10]

    As the organism develops, it can come to possess organs that have different sets of chromosomes. For example, the chimera may have a liver composed of cells with one set of chromosomes and have a kidney composed of cells with a second set of chromosomes. This has occurred in humans, and at one time was thought to be extremely rare although more recent evidence suggests that this is not the case. [1] [11]

    This is particularly true for the marmoset. Recent research shows most marmosets are chimeras, sharing DNA with their fraternal twins. [12] 95% of marmoset fraternal twins trade blood through chorionic fusions, making them hematopoietic chimeras. [13] [14]

    Most chimeras will go through life without realizing they are chimeras. The difference in phenotypes may be subtle (e.g., having a hitchhiker's thumb and a straight thumb, eyes of slightly different colors, differential hair growth on opposite sides of the body, etc.) or completely undetectable. Chimeras may also show, under a certain spectrum of UV light, distinctive marks on the back resembling that of arrow points pointing downwards from the shoulders down to the lower back this is one expression of pigment unevenness called Blaschko's lines. [15]

    Affected persons may be identified by the finding of two populations of red cells or, if the zygotes are of opposite sex, ambiguous genitalia and intersex alone or in combination such persons sometimes also have patchy skin, hair, or eye pigmentation (heterochromia). If the blastocysts are of opposite sex, genitals of both sexes may be formed: either ovary and testis, or combined ovotestes, in one rare form of intersex, a condition previously known as true hermaphroditism. [ citation needed ]

    Note that the frequency of this condition does not indicate the true prevalence of chimerism. Most chimeras composed of both male and female cells probably do not have an intersex condition, as might be expected if the two cell populations were evenly blended throughout the body. Often, most or all of the cells of a single cell type will be composed of a single cell line, i.e. the blood may be composed predominantly of one cell line, and the internal organs of the other cell line. Genitalia produce the hormones responsible for other sex characteristics.

    Natural chimeras are almost never detected unless they exhibit abnormalities such as male/female or hermaphrodite characteristics or uneven skin pigmentation. The most noticeable are some male tortoiseshell cats and calico cats (although most male tortoiseshells have an extra X chromosome responsible for the colouration) or animals with ambiguous sex organs. [ citation needed ]

    The existence of chimerism is problematic for DNA testing, a fact with implications for family and criminal law. The Lydia Fairchild case, for example, was brought to court after DNA testing apparently showed that her children could not be hers. Fraud charges were filed against her and her custody of her children was challenged. The charge against her was dismissed when it became clear that Lydia was a chimera, with the matching DNA being found in her cervical tissue. [ citation needed ] Another case was that of Karen Keegan, who was also suspected (initially) of not being her children's biological mother, after DNA tests on her adult sons for a kidney transplant she needed, seemed to show she was not their mother. [1] [16]

    The tetragametic state has important implications for organ or stem cell transplantation. Chimeras typically have immunologic tolerance to both cell lines. [ citation needed ]

    Microchimerism Edit

    Microchimerism is the presence of a small number of cells that are genetically distinct from those of the host individual. Most people are born with a few cells genetically identical to their mothers' and the proportion of these cells goes down in healthy individuals as they get older. People who retain higher numbers of cells genetically identical to their mother's have been observed to have higher rates of some autoimmune diseases, presumably because the immune system is responsible for destroying these cells and a common immune defect prevents it from doing so and also causes autoimmune problems. The higher rates of autoimmune diseases due to the presence of maternally-derived cells is why in a 2010 study of a 40-year-old man with scleroderma-like disease (an autoimmune rheumatic disease), the female cells detected in his blood stream via FISH (fluorescence in situ hybridization) were thought to be maternally-derived. However, his form of microchimerism was found to be due to a vanished twin, and it is unknown whether microchimerism from a vanished twin might predispose individuals to autoimmune diseases as well. [17] Mothers often also have a few cells genetically identical to those of their children, and some people also have some cells genetically identical to those of their siblings (maternal siblings only, since these cells are passed to them because their mother retained them). [ citation needed ]

    Symbiotic chimerism in anglerfish Edit

    Chimerism occurs naturally in adult Ceratioid anglerfish and is in fact a natural and essential part of their life cycle. Once the male achieves adulthood, it begins its search for a female. Using strong olfactory (or smell) receptors, the male searches until it locates a female anglerfish. The male, less than an inch in length, bites into her skin and releases an enzyme that digests the skin of both his mouth and her body, fusing the pair down to the blood-vessel level. While this attachment has become necessary for the male's survival, it will eventually consume him, as both anglerfish fuse into a single hermaphroditic individual. Sometimes in this process, more than one male will attach to a single female as a symbiote. In this case, they will all be consumed into the body of the larger female angler. Once fused to a female, the males will reach sexual maturity, developing large testicles as their other organs atrophy. This process allows for sperm to be in constant supply when the female produces an egg, so that the chimeric fish is able to have a greater number of offspring. [18]

    Germline chimerism Edit

    Germline chimerism occurs when the germ cells (for example, sperm and egg cells) of an organism are not genetically identical to its own. It has been recently discovered that marmosets can carry the reproductive cells of their (fraternal) twin siblings due to placental fusion during development. (Marmosets almost always give birth to fraternal twins.) [12] [19] [20]

    Artificial chimerism Edit

    Artificial chimerism falls under the artificial category in which a chimera can exist. An individual that falls under this classification possesses two different sets of genetic pedigrees: one that was inherited genetically at the time of the formation of the human embryo and the other that was intentionally introduced through a medical procedure known as transplantation. [21] Specific types of transplants that could induce this condition include bone marrow transplants and organ transplants, as the recipient's body essentially works to permanently incorporate the new blood stem cells into it.

    An example of artificial chimerism in animals are the quail-chick chimeras. By utilizing transplantation and ablation in the chick embryo stage, the neural tube and the neural crest cells of the chick were ablated, and replaced with the same parts from a quail. [22] Once hatched, the quail feathers were visibly apparent around the wing area, whereas the rest of the chick's body was made of its own chicken cells.

    Humans Edit

    Chimerism has been documented in humans in several instances.

    • The Dutch sprinter Foekje Dillema was expelled from the 1950 national team after she refused a mandatory sex test in July 1950 later investigations revealed a Y-chromosome in her body cells, and the analysis showed that she was probably a 46,XX/46,XY mosaic female. [23]
    • In 1953 a human chimera was reported in the British Medical Journal. A woman was found to have blood containing two different blood types. Apparently this resulted from her twin brother's cells living in her body. [24] A 1996 study found that such blood group chimerism is not rare. [25]
    • Another report of a human chimera was published in 1998, where a male human had some partially developed female organs due to chimerism. He had been conceived by in-vitro fertilization. [4]
    • In 2002, Lydia Fairchild was denied public assistance in Washington state when DNA evidence appeared to show that she was not the mother of her children. A lawyer for the prosecution heard of a human chimera in New England, Karen Keegan, and suggested the possibility to the defense, who were able to show that Fairchild, too, was a chimera with two sets of DNA, and that one of those sets could have been the mother of the children. [26]
    • In 2002, an article in the New England Journal of Medicine describes a woman in whom tetragametic chimerism was unexpectedly identified after undergoing preparations for kidney transplant that required the patient and her immediate family to undergo histocompatibility testing, the result of which suggested that she was not the biologic mother of two of her three children. [27]
    • In 2009, singer Taylor Muhl discovered that what was always thought to be a large birthmark on her torso was actually caused by chimerism.
    • In 2017, a human-pig chimera was reported to have been created the chimera was also reported to have 0.001% human cells, with the balance being pig. [28][29][30]
    • In 2021, a human-monkey chimera was created as a joint project between the Salk Institute in the USA and Kunming University in China and published in the Journal, Cell. [31] This involved injecting human stem cells into monkey embryos. The embryos were only allowed to grow for a few days but the study demonstrated that some of these embryos still had human stem cells surviving at the end of the experiments. Because humans are more closely related to monkeys than other animals, it means there is more chance of the chimeric embryos surviving for longer periods so that organs can develop. The project has opened up possibilities into organ transplantation as well as ethical concerns particularly concerning human brain development in primates. [32]

    Hermaphrodites Edit

    • Debate exists surrounding true hermaphrodites in regards to a hypothetical scenario in which it could be possible for a human to self-fertilize. If a human chimera is formed from a male and female zygote fusing into a single embryo, giving an individual functional gonadal tissue of both types, such a self-fertilization is feasible. Indeed, it is known to occur in non-human species where hermaphroditic animals are common. However, no such case of functional self-fertilization has ever been documented in humans. [33]

    Bone marrow recipients Edit

    • Several cases of unusual chimera phenomena have been reported in bone marrow recipients.
      • In 2019, the blood and seminal fluid of a man in Reno, Nevada (who had undergone a vasectomy), exhibited only the genetic content of his bone marrow donor. Swabs from his lips, cheek and tongue showed mixed DNA content. [34]
      • The DNA content of semen from an assault case in 2004 matched that of a man who had been in prison at the time of the assault, but who had been a bone marrow donor for his brother, who was later determined to have committed the crime. [34][35][36]
      • In 2008, a man was killed in a traffic accident that occurred in Seoul, South Korea. In order to identify him, his DNA was analyzed. Results revealed that the DNA of his blood, along with some of his organs, appeared to show that he was female. It was later determined that he had received a bone marrow transplant from his daughter. [34]

      Chimera Identification Edit

      Chimerism is so rare that there have only been 100 confirmed cases in humans. [37] However, this may be due to the fact that humans might not be aware that they have this condition to begin with. There are usually no signs or symptoms for chimerism other than a few physical symptoms such as hyper-pigmentation, hypo-pigmentation, or possessing two different colored eyes. However, these signs do not necessarily mean an individual is a chimera and should only be seen as possible symptoms. Again, forensic investigation or curiosity over a failed maternity/paternity DNA test usually leads to the accidental discovery of this condition. By simply undergoing a DNA test, which usually consists of either a swift cheek swab or a blood test, the discovery of the once unknown second genome is made, therefore identifying that individual as a chimera. [38]

      Research Edit

      The first known primate chimeras are the rhesus monkey twins, Roku and Hex, each having six genomes. They were created by mixing cells from totipotent four cell blastocysts although the cells never fused, they worked together to form organs. It was discovered that one of these primates, Roku, was a sexual chimera as four percent of Roku's blood cells contained two x chromosomes. [13]

      A major milestone in chimera experimentation occurred in 1984 when a chimeric sheep–goat was produced by combining embryos from a goat and a sheep, and survived to adulthood. [39]

      In August 2003, researchers at the Shanghai Second Medical University in China reported that they had successfully fused human skin cells and rabbit ova to create the first human chimeric embryos. The embryos were allowed to develop for several days in a laboratory setting, and then destroyed to harvest the resulting stem cells. [40] In 2007, scientists at the University of Nevada School of Medicine created a sheep whose blood contained 15% human cells and 85% sheep cells. [41]

      On January 22, 2019 the National Society of Genetic Counselors released an article — Chimerism Explained: How One Person Can Unknowingly Have Two Sets of DNA, where they state “Tetragametic Chimerism, where a twin pregnancy evolves into one child, is currently believed to be one of the rarer forms. However, we know that 20 to 30 percent of singleton pregnancies were originally a twin or a multiple pregnancy. Due to this statistic, it is quite possible that tetragametic chimerism is more common than current data implies”. [42]

      Sponges Edit

      Chimerism has been found in some species of marine sponges. [43] Four distinct genotypes have been found in a single individual, and there is potential for even greater genetic heterogeneity. Each genotype functions independently in terms of reproduction, but the different intra-organism genotypes behave as a single large individual in terms of ecological responses like growth. [43]

      Mice Edit

      Chimeric mice are important animals in biological research, as they allow for the investigation of a variety of biological questions in an animal that has two distinct genetic pools within it. These include insights into problems such as the tissue specific requirements of a gene, cell lineage, and cell potential. The general methods for creating chimeric mice can be summarized either by injection or aggregation of embryonic cells from different origins. The first chimeric mouse was made by Beatrice Mintz in the 1960s through the aggregation of eight-cell-stage embryos. [44] Injection on the other hand was pioneered by Richard Gardner and Ralph Brinster who injected cells into blastocysts to create chimeric mice with germ lines fully derived from injected embryonic stem cells (ES cells). [45] Chimeras can be derived from mouse embryos that have not yet implanted in the uterus as well as from implanted embryos. ES cells from the inner cell mass of an implanted blastocyst can contribute to all cell lineages of a mouse including the germ line. ES cells are a useful tool in chimeras because genes can be mutated in them through the use of homologous recombination, thus allowing gene targeting. Since this discovery occurred in 1988, ES cells have become a key tool in the generation of specific chimeric mice. [46]

      Underlying biology Edit

      The ability to make mouse chimeras comes from an understanding of early mouse development. Between the stages of fertilization of the egg and the implantation of a blastocyst into the uterus, different parts of the mouse embryo retain the ability to give rise to a variety of cell lineages. Once the embryo has reached the blastocyst stage, it is composed of several parts, mainly the trophectoderm, the inner cell mass, and the primitive endoderm. Each of these parts of the blastocyst gives rise to different parts of the embryo the inner cell mass gives rise to the embryo proper, while the trophectoderm and primitive endoderm give rise to extra embryonic structures that support growth of the embryo. [47] Two- to eight-cell-stage embryos are competent for making chimeras, since at these stages of development, the cells in the embryos are not yet committed to give rise to any particular cell lineage, and could give rise to the inner cell mass or the trophectoderm. In the case where two diploid eight-cell-stage embryos are used to make a chimera, chimerism can be later found in the epiblast, primitive endoderm, and trophectoderm of the mouse blastocyst. [48] [49]

      It is possible to dissect the embryo at other stages so as to accordingly give rise to one lineage of cells from an embryo selectively and not the other. For example, subsets of blastomeres can be used to give rise to chimera with specified cell lineage from one embryo. The Inner Cell Mass of a diploid blastocyst, for example, can be used to make a chimera with another blastocyst of eight-cell diploid embryo the cells taken from the inner cell mass will give rise to the primitive endoderm and to the epiblast in the chimera mouse. [50] From this knowledge, ES cell contributions to chimeras have been developed. ES cells can be used in combination with eight-cell-and two-cell-stage embryos to make chimeras and exclusively give rise to the embryo proper. Embryos that are to be used in chimeras can be further genetically altered in order to specifically contribute to only one part of chimera. An example is the chimera built off of ES cells and tetraploid embryos, which are artificially made by electrofusion of two two-cell diploid embryos. The tetraploid embryo will exclusively give rise to the trophectoderm and primitive endoderm in the chimera. [51] [52]

      Methods of production Edit

      There are a variety of combinations that can give rise to a successful chimera mouse and – according to the goal of the experiment – an appropriate cell and embryo combination can be picked they are generally but not limited to diploid embryo and ES cells, diploid embryo and diploid embryo, ES cell and tetraploid embryo, diploid embryo and tetraploid embryo, ES cells and ES cells. The combination of embryonic stem cell and diploid embryo is a common technique used for the making of chimeric mice, since gene targeting can be done in the embryonic stem cell. These kinds of chimeras can be made through either aggregation of stem cells and the diploid embryo or injection of the stem cells into the diploid embryo. If embryonic stem cells are to be used for gene targeting to make a chimera, the following procedure is common: a construct for homologous recombination for the gene targeted will be introduced into cultured mouse embryonic stem cells from the donor mouse, by way of electroporation cells positive for the recombination event will have antibiotic resistance, provided by the insertion cassette used in the gene targeting and be able to be positively selected for. [53] [54] ES cells with the correct targeted gene are then injected into a diploid host mouse blastocyst. Then, these injected blastocysts are implanted into a pseudo pregnant female surrogate mouse, which will bring the embryos to term and give birth to a mouse whose germline is derived from the donor mouse's ES cells. [55] This same procedure can be achieved through aggregation of ES cells and diploid embryos, diploid embryos are cultured in aggregation plates in wells where single embryos can fit, to these wells ES cells are added the aggregates are cultured until a single embryo is formed and has progressed to the blastocyst stage, and can then be transferred to the surrogate mouse. [56]


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