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11.5: Transgenic Animals - Biology

11.5: Transgenic Animals - Biology


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A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. In addition to the gene itself, the DNA usually includes other sequences to enable it to be incorporated into the DNA of the host and to be expressed correctly by the cells of the host. Transgenic sheep and goats have been produced that express foreign proteins in their milk. Transgenic chickens are now able to synthesize human proteins in the "white" of their eggs. These animals should eventually prove to be valuable sources of proteins for human therapy.

Note

In July 2000, researchers from the team that produced Dolly reported success in producing transgenic lambs in which the transgene had been inserted at a specific site in the genome and functioned well.

Transgenic mice have provided the tools for exploring many biological questions.

Example

Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-manipulated model for studying the disease. However, transgenic mice expressing the human gene for the polio virus receptor

  • can be infected by polio virus and even
  • develop paralysis and other pathological changes characteristic of the disease in humans.

Two methods of producing transgenic mice are widely used:

  • transforming embryonic stem cells (ES cells) growing in tissue culture with the desired DNA
  • injecting the desired gene into the pronucleus of a fertilized mouse egg

Fig.11.4.1 Methods to produce Transgenic mice

The Embryonic Stem Cell Method - Method 1

Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts. They can be grown in culture and retain their full potential to produce all the cells of the mature animal, including its gametes.

1. Make your DNA

Using recombinant DNA methods, build molecules of DNA containing

  • the gene you desire (e.g., the insulin gene)
  • vector DNA to enable the molecules to be inserted into host DNA molecules
  • promoter and enhancer sequences to enable the gene to be expressed by host cells

2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.

3. Select for successfully transformed cells

4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.

5. Embryo transfer

  • Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male). The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
  • Transfer the embryos into her uterus.
  • Hope that they implant successfully and develop into healthy pups (no more than one-third will).

6. Test her offspring

  • Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No more than 10–20% will have it, and they will be heterozygous for the gene.

7. Establish a transgenic strain

  • Mate two heterozygous mice and screen their offspring for the 1 in 4 that will be homozygous for the transgene.
  • Mating these will found the transgenic strain.

The Pronucleus Method - Method 2

1. Prepare your DNA as in Method 1

2. Transform fertilized eggs

  • Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
  • Inject the male pronucleus with your DNA.
  • When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide by mitosis to form a 2-cell embryo.

3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.

Example

This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a normal littermate (left). The giant mouse developed from a fertilized egg transformed with a recombinant DNA molecule containing:

  • the gene for human growth hormone
  • a strong mouse gene promoter

The levels of growth hormone in the serum of some of the transgenic mice were several hundred times higher than in control mice.

Random vs. Targeted Gene Insertion

The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a particular gene, it is possible to design vectors that replace that gene. The replacement gene can be one that

  • restores function in a mutant animal or
  • knocks out the function of a particular locus.

In either case, targeted gene insertion requires

  • the desired gene
  • neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives, like the drug G418, which is lethal to mammalian cells
  • tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside analog ganciclovir. DNA polymerase fails to discriminate against the resulting nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills cells that contain the tk gene

Step 1

Treat culture of ES cells with preparation of vector DNA.

Results:

  • Most cells fail to take up the vector; these cells will be killed if exposed to G418.
  • In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but killed by gancyclovir.
  • In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the vector find the homologous sequences in the host genome, and the region between these homologous sequences replaces the equivalent region in the host DNA.

Step 2

Culture the mixture of cells in medium containing both G418 and ganciclovir.

  • The cells (the majority) that failed to take up the vector are killed by G418.
  • The cells in which the vector was inserted randomly are killed by gancyclovir (because they contain the tk gene).
  • This leaves a population of cells transformed by homologous recombination (enriched several thousand fold).

Step 3

Inject these into the inner cell mass of mouse blastocysts.

Knockout Mice: What do they teach us?

If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out"). Knockout mice are valuable tools for discovering the function(s) of genes for which mutant strains were not previously available. Two generalizations have emerged from examining knockout mice:

  • Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
  • Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.

Tissue-Specific Knockout Mice

While "housekeeping" genes are expressed in all types of cells at all stages of development, other genes are normally expressed in only certain types of cells when turned on by the appropriate signals (e.g. the arrival of a hormone).

To study such genes, one might expect that the methods described above would work. However, it turns out that genes that are only expressed in certain adult tissues may nonetheless be vital during embryonic development. In such cases, the animals do not survive long enough for their knockout gene to be studied. Fortunately, there are now techniques with which transgenic mice can be made where a particular gene gets knocked out in only one type of cell.

The Cre/loxP System

One of the bacteriophages that infects E. coli, called P1, produces an enzyme — designated Cre — that cuts its DNA into lengths suitable for packaging into fresh virus particles. Cre cuts the viral DNA wherever it encounters a pair of sequences designated loxP. All the DNA between the two loxP sites is removed, and the remaining DNA ligated together again (so the enzyme is a recombinase). Using "Method 1" above, mice can be made transgenic for

  • the gene encoding Cre attached to a promoter that will be activated only when it is bound by the same transcription factors that turn on the other genes required for the unique function(s) of that type of cell;
  • a "target" gene, the one whose function is to be studied, flanked by loxP sequences.

In the adult animal,

  • those cells that
    • receive signals (e.g., the arrival of a hormone or cytokine)
    • to turn on production of the transcription factors needed
    • to activate the promoters of the genes whose products are needed by that particular kind of cell
    will also turn on transcription of the Cre gene. Its protein will then remove the "target" gene under study.
  • All other cells will lack the transcription factors needed to bind to the Cre promoter (and/or any enhancers) so the target gene remains intact.

The result: a mouse with a particular gene knocked out in only certain cells.

Knock-in Mice

The Cre/loxP system can also be used to

  • remove DNA sequences that block gene transcription. The "target" gene can then be turned on in certain cells or at certain times as the experimenter wishes.
  • replace one of the mouse's own genes with a new gene that the investigator wishes to study.

Such transgenic mice are called "knock-in" mice.

Transgenic Sheep and Goats

Until recently, the transgenes introduced into sheep inserted randomly in the genome and often worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals expressed large quantities of the human protein in their milk.

This is how it was done.

Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that contained these segments of DNA:

  1. 2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence in humans causes the inherited disease osteogenesis imperfecta.)

    This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would expect the gene to be easily accessible in the chromatin.

  2. A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector.
  3. The human gene encoding alpha1-antitrypsin.

    Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or absence produces the disease Alpha1-Antitrypsin Deficiency (A1AD or Alpha1). The main symptoms are damage to the lungs (and sometimes to the liver).

  4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in milk-producing cells.
  5. Binding sites for ribosomes for efficient translation of the beta-lactoglobulin mRNAs.

Successfully-transformed cells were then

  • Fused with enucleated sheep eggs and implanted in the uterus of a ewe (female sheep)
  • Several embryos survived until their birth, and two young lambs lived over a year.
  • When treated with hormones, these two lambs secreted milk containing large amounts of alpha1-antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the transgene).

On June 18, 2003, the company doing this work abandoned it because of the great expense of building a facility for purifying the protein from sheep's milk. Purification is important because even when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins that remain.

However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the milk of transgenic goats.

Transgenic Chickens

Chickens grow faster than sheep and goats and large numbers can be grown in close quarters. They also synthesize several grams of protein in the "white" of their eggs.Two methods have succeeded in producing chickens carrying and expressing foreign genes.

  • Infecting embryos with a viral vector carrying
    • the human gene for a therapeutic protein
    • promoter sequences that will respond to the signals for making proteins (e.g. lysozyme) in egg white
  • Transforming rooster sperm with a human gene and the appropriate promoters and checking for any transgenic offspring.

Preliminary results from both methods indicate that it may be possible for chickens to produce as much as 0.1 g of human protein in each egg that they lay.

Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.

Transgenic Pigs

Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may someday be able to produce transgenic pigs that can serve as a source of transplanted organs for humans.

Transgenic Primates

In the 28 May 2009 issue of Nature, Japanese scientists reported success in creating transgenic marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the germline and passed on to the animal's offspring. The hope is that these transgenic animals will provide the best model yet for studying human disease and possible therapies.


3 Important Examples of Transgenic Animal | Genetics

The following points highlight the three important examples of transgenic animal. The examples are: 1. Cloning Dolly 2. Transgenic Mice 3. Reporter System.

Example # 1. Cloning Dolly:

In February 1996, Ian Wilmut and co-workers from the Roslin Institute and PPL Therapeutics, both in Edinburgh, Scotland, reported in Nature journal that they had successfully cloned a sheep from a cell taken from the udder of a six-year old ewe.

The cloned lamb was named Dolly. In the past, genetically identical animal embi70s had been created only with amphibian cells, and those created from adult nuclei had never successfully reached adulthood. Cloning in which the nuclei came from fetal cells or cells from cell lines had been successful before in mammals. The word clone means to create a genetically identical copy.

To clone an animal, it is necessary to begin with an egg, the only cell known to initiate and support development. In order to clone an individual, it is necessary to get an egg without a nucleus and then to transplant in it a nucleus of known origin.

Techniques for nuclear transplantation had been worked out with frogs and toads in the 1950s. The Scottish scientists succeeded in obtaining sheep eggs, enucleating them (removing their nuclei) and then transferring in donor nuclei by fusing the donor cells and the enucleated eggs with an electric pulse.

The electric pulse also initiated development of the egg. Although only one pregnancy of the twenty nine initiated was successful, the lamb that was born seemed normal in every respect since it had produced offspring.

Others had tried this type of experiments with many types of animals, including mice. They were not successful for numerous reasons. The most likely explanation for the recent success, according to scientists, is that the donor cells were kept in a non-growth phase for several days, which may have synchronized them with the oocyte.

Thus the nucleus and the oocyte were at the same stage of cell cycle and thus compatible. Other reorganization that had to take place in the donor chromosomes are not really known for certain but one thing is clear: the nucleus of an adult cell in the sheep has all of the genetic material needed to support normal growth and development of the egg. The work has since been repeated with goats, cattle and mice.

There are various ramifications to the success of this work:

1. Mammal cloning could becomes a routine procedure. This would allow us to study mammalian development and to replicate genetically identical individuals, particularly transgenic animals that would have particular genome of value.

2. We can also use these techniques to study aging, since an “old” nucleus in initiating development of a new organism.

3. Also, of interest is the interaction of a particular genome with a particular cytoplasm, since the cytoplasm contains not only the material needed for early development, but also cell organelles, including mitochondria that have their own genetic material.

Example # 2. Transgenic Mice:

Animal cells, like the protoplasts of plant cells, can take up foreign chromosomes or DNA directly from the environment with a very low efficiency (in the presence of calcium phosphate). Directly injecting the DNA greatly improves the efficiency.

For example, transgenic mice are now routinely prepared by injecting DNA either into oocytes or one or two- celled embryos obtained from female mice after appropriate hormonal treatment.

After injection of about 2 picoliters (2 X 10 -12 liters) of cloned DNA, the cells are reimplanted into the uteruses of receptive female hosts. In about 15% of these injections, the foreign DNA incorporates into the embryo.

Transgenic animals are used to study the expression and control of foreign eukaryotic genes. In 1988, a transgenic mouse prone to cancer was first genetically engineered animal to be patented. Thus mouse provides an excellent model for studying cancer. (A controversy arose as to whether engineered higher organisms should be patentable currently they are).

Mice have already been successfully transfected with a rat growth-hormone gene, and transgenic sheep have been produced that express the gene for a human clotting factor. The latest recombinant DNA dispute arose, from cloning of sheep in 1997.

The transfection can also be mediated by retroviruses (RNA viruses containing the gene for reverse transcriptase). For example, a retroviral vector was ritrodused and repaired human white blood cell lacking the enzyme adenosine deaminase.

A retrovirus responsible for a form of leukemia in rodents, the Moloney Murine leukemia viruses was engineered so that all the virus genes were removed and replaced with an antibiotic marker (neomycin resistance) and the human adenosine deaminase gene.

The virus binds to the cell surface and is taken into the cell, its RNA is converted to DNA by reverse transcription and the DNA is incorporated into one of the cells, chromosomes. It is not possible for the highly modified virus to attack and damage the cells.

Example # 3. Reporter System:

Two reporter systems are used to indicate that a transfection experiment was successful. Plants can be transfected with the Ti plasmids of Agrobacterium tumefaciens. When a plant is infected with A. tumefaciens containing the Ti plasmid, a crown gall tumor is induced transferring the T- DNA region.

Those cells transfected with the T-DNA are induced to grow as well as to produce opines that the bacteria feed on. Much recent research has concentrated on engineering Ti plasmids to contain other genes that are also transferred to the host plants during infection, creating transgenic plants. One series of experiments have been especially charming.

Tobacco plants have been transinfected by Ti plasmids containing the luciferase gene from fireflies. The product of this gene catalyzes the ATP- dependent oxidation of luciferin, which emits light. When a transfected plant is watered with luciferin, it glows like a firefly. The value of these experiments is not the production of glowing plants but rather the use of the glow to “report” the action of specific genes.

In further experiments, the promoters and enhancers of certain genes were attached to the luciferase gene. As a result, luciferase would only be produced when these promoters were activated thus, the glowing areas of the plant show where the transfected gene is active.

One of the more recent reported systems developed uses a gene from jellyfish that produces green fluorescent protein. The value of this system is that it “reports” when ultra-violet light falls on it, rather than it requiring an addition, as in the luciferase system (see Tamarin, 2002).


Transgenic Animals

Transgenic Mice and other Animals. One of the first reports of transgenic animals published in December, 1982, involved the transfer of the growth hormone (GH) gene (from rat) fused to the promoter for the mouse metallothionein 1 (MT) gene. Since then many transgenic animals, including those in cattle, sheep, goats, pigs, rabbits, chickens and fish have been produced and will be utilized in future for a variety of purposes including

(i) their efficiency in utilizing feed

(ii) ability to give leaner meat,

(iii) their ability to grow to marketable size sooner and

(iv) their resistance to certain diseases.

More than this recently efforts are being made to use transgenic animals as living bioreactors. Transgenic animals produced for this purpose will secrete valuable recombinant proteins and pharmaceuticals into their milk, blood and urine which can be used for extraction of these drugs. This new possibility of manufacturing drugs through transgenic animals is often described as ‘molecular farming’ or ‘molecular pharming’.

Gene abbreviations – ALV = avian leukosis virus alAT = Al anti trypsin BPV = bovine papilloma virus Eu = immunoglobulin heavy chain FIX = factor IX: GH = growth hormone GRF= growth releasing factor, β Gal = β galactosidase hygo = hygromycin BLG = β – lactoglobulin MT= metallothionein MLV= moloney murine leukemia virus c-myc = a cellular proto oncogene REV = reticuloendotheliosis PRL = prolactin SV= SV = SV 40 TK = thymidine kinase AFP = antifreeze protein tPA = human tissue-type plasminogen activator.

Although initially many experiments leading to the production of transgenic animals did not give commercially attractive results, success has been obtained in some recent cases. Some of these cases will be discussed here.

Transgenic Goats.

Transgenic goats were successfully produced recently by groups headed by John McPherson and Karl Ebert, both from the USA. These transgenic goats expressed a heterologous protein (a variant of human tissue-type plasminogen activator = LAtPA) in their milk. This protein is used for dissolving blood clots ie., for the treatment of coronary thrombosis. A cDNA representing LAtPA was linked with either the murine whey acid promoter (WAP) or a β casein promoter in an expression vector and used for injecting early embryos obtained surgically from the oviducts of superovulated dairy goats. These injected embryos were either immediately transferred to the oviducts of recipient females (surrogate mothers) or cultured for 72 hours (blocked at 8-16 cell stage), before transfer to the uterus or recipient females of 29 offsprings from 36 recipients, one male and one female contained the transgene. The transgenic female delivered five offsprings, one of which was transgenic showing expression of LAtPA at a low level of few milligrams per litre of milk. In another case of a transgenic goat, few grams of LAtPA per litre of milk could be obtained. At this concentration, the dairy goat may become an economically viable bioreactor for human pharmaceuticals.

Transgenic Cows.

In most earlier attempts (in Canada) for the production of transgenic cows, embryos or fertilized oocytes produced in vivo were utilized. Fertilized oocytes or proembryos were surgically terrieved from superovulated and artificially inseminated cows. Microinjected zygotes were than transferred by surgery either directly into the oviduct of recipient cows or into temporary hosts like sheep or rabbits. In view of two surgical operations, this method is labour intensive and more expensive.

In ‘the Netherland’, recently (1991) a technique has been developed for in vitro embryo production. In this new procedure, oocytes obtained from the ovaries of slaughter house cows, were matured and fertilized in vitro. Their pronuclei were microinjected with a construct containing a bovine alpha – Sl casei promoter (bovine = ox) driving a cDNA encoding the antibacterial human iron-binding protein, ‘lactoferrin’. The embryos were cultured to the morula/ blastula stage and then non-surgically transferred to recipient females. Two of the 19 calves born from 103 transferred zygotes were transgenic (one male and other female). This procedure may facilitate the use of cows as bioreactors at the commercial level.

Transgenic Fish.

Alilempis to produce transgenic fish started in 1985 and some encouraging results have been obtained. The genes that have been introduced by microinjection in fish include the following:

(i) human or rat gene for growth hormone,

(ii) chicken gene for delta-crystalline protein,

(iii) E. coli gene for β-galactosidase,

(iv) E.coli gene for neomycin resistance,

(v) winter flounder gene for antifreeze protein (flounder-flat fish),

(vi) rainbow trout gene for growth hormone.

The technique of microinjection has been successfully used to generate transgenic in many species such as common carp, catfish, goldfish, loach, medaka, salmon, Tilapia, rainbow trout and zebrafish. In other animals (e.g., mice, cows, pigs, sheep and rabbits), usually direct microinjection of cloned DNA into male pronuclei of fertilized eggs has proved very successful, but in most fish species studied so far, pronuclei cannot be easily visualized (except in medaka), so that the DNA needs to be injected into the cytoplasm.

Eggs and sperms from mature individuals are collected and placed into a separate dry container. Fertilization is initiated by adding water and sperm to eggs, with gentle stirring to facilitate the fertilization process. Egg shells are hardened in water. About 10° to 10° molecules of linearized DNA in a volume of 20 ml or less are microinjected into each egg (1-4 cells stage) within the first few hours after fertilization. Following microinjection, eggs are incubated in appropriate hatching trays, and dead embryos are removed daily.

Since in fish, fertilization is external, in vitro culturing of embryos and their subsequent transfer into foster mothers (required in mammalian systems) is not required. Further, the injection into the cytoplasm is not as harmful as that into the nucleus, so that the survival rate in fish is much higher (35% to 80%).

Human growth hormone gene transferred to transgenic fish allowed growth that was twice the size for their corresponding non-transgenic fish (goldfish, rainbow trout, salmon). Similarly, antifreeze protein (AFP) gene was transferred in several cases and its expression was studied in transgenic salmon. It was shown that the level of AFP gene expression is still too low to provide protection against freeze. There is also a report (in 1991) of the production of transgenic zebrafish from an Indian Laboratory (Madurai Kamraj University). In this attempt, a plasmid containing rat growth hormone gene was microinjected into fertilized zebrafish eggs, and its presence confirmed in adult fish.

Transgenic Pigs.

The efficiency of the production of transgenic pigs is still very low compared to that of the production of transgenic mice. In mice, 2.5% to 6% of the microinjected eggs developed into transgenic mice, but in pigs this frequency was as low as 0.6% even when as many as 7000 eggs were injected. Despite this low frequency, transgenic pigs carrying growth hormone (GH) gene from bovine (of ‘ox’ origin) of human, and sheep globin gene have been produced (by V.G. Purse) at Agriculture Research Service, Beltsville, USA. The pigs carrying hGH gene showed different levels of expression and only 66% of these animals showed detectable levels of hGH and bGH in their plasma. The animals grew a little faster but did not become large similarly pigs with sheep globin gene did not show any expression of the transgene for unknown reasons. In these transgenic pigs, however, a modest increase of 10-15% in daily weight and 16-18% in feed efficiency was observed, which is though lower to those in mice, but are comparable to those obtained due to daily injection with pig growth hormone. It was also observed that there was a marked reduction in the subcutaneous fat in some of these transgenic pigs suggesting the possibility of producing leaner meat with lower fat content. These results may have a significant impact on the 9.5 billion dollar annual pig industry in USA.

It is also reported that a long term elevation of growth hormone was generally detrimental to health. The pigs had incidence of gastric ulcers, arthritis and several other diseases. Therefore, techniques will have to be developed to manipulate better the transgene expression by a variety of methods (e.g., changing genetic background or modifying the husbandry regiment).

Transgenic Sheep.

The rate of transgenesis in sheep is very low (0.1 to 0.2%). This can be improved, if only transgenic viable embryos (after necessary checking) are transferred to surrogate ewes (female sheep). Embryos at 8-16 cell stage can be split into two parts, one for continued culture and the other for detection of integrated genes using polymerase chain reaction (PCR). Although microinjection is the most common method for DNA delivery, gene targeting may be increasingly used in the future. In this approach, embryonic stem (ES) cells in culture are transferred with a vector that targets the gene to a particular site by homologous recombination (as discussed above). The technique, though successfully used in mice, has yet to be applied to sheep, where ES cells will have to be isolated first.

The first reports of transgenic sheep were published by J.P. Simons (1988) of Edinburgh. Two transgenic ewes were produced, each carrying about 10 copies of human anti-hemophilic factor IX gene (cDNA) fused with the 10.5 kb BLG gene (BLG = β -lactoglobulin). BLG gene was used, because it is necessary for specific expression of gene in mammary glands. Consequently, the gene had a tissue specific expression and ewes secreted human factor IX (or alpha-1 antitrypsin) into their milk this human factor IX is active, even though the expression of transgenic is low. The transgenic ewes were born in early summer of 1986 and were successfully mated same year in December. In 1987, each ewes gave birth to a single lamb. Each lamb inherited BLG-F IX transgene and secreted factor IX in the milk. This programme of the production of transgenic animals by J.P. Simons at Edinburgh was funded by Pharmaceutical Proteins Ltd. (Cambridge, UK) due to its commercial appeal.

In another report (published in 1991), also from Edinburgh, five transgenic sheep were produced (Alan Colman and colleagues). In all these cases, transgene involved fusion of the ovine β-lactoglobulin gene promoter fused to the human aq, antitrypsin (h a 1 AT) gene. Four of these animals were female and one male. In one female the protein hATh a 1 AT reached a level of 35 grams per litre of milk. The protein purified from milk had a biological activity indistinguishable from human plasma derived antitrypsin. The deficiency for h a 1 AT leads to a lethal disease emphysema, (BC-22), which is a common hereditary disorder among caucasian males of European descent. Therefore any strategy giving high yield of this protein economically will be most welcome. In view of this, transgenic sheep with h a 1 AT gene will prove very useful as a bioreactor.

Recombinant DNA technique can also be used to increase the ability of sheep for wool growth. For this purpose, genes essential for synthesis of some important amino acids found in keratin proteins of wool, have been cloned and introduced in embryos to produce transgenic sheep. For instance, genes (cysE and cysM) for two enzymes (serine acetyl transferase = SAT and O-acetylserine sulphydryase = OAS), involved in cystein biosynthesis, were isolated from bacteria and cloned in a vector. These genes were introduced in sheep cells, ultimately leading to the production of transgenic sheep, where these genes are expressed. Growth hormone (GH) genes have also been introduced and can be used to promote body weight. Other genes involved in wool production have also been cloned and well be used for transgenesis, thus increasing the potential of wool production through genetic engineering.


Transgenic Animals

The growth in the field of molecular biology and biotechnology is due to the intensive research using transgenic animals. However, there are many ethical issues regarding the use of transgenic animals, because, the genome of transgenic animals is deliberately modified. The first transgenic animals called chimeric mice were created by combining two cells taken from two different embryos of different strains. This gave rise to a single embryo, which was implanted into a surrogate mother to give birth to a chimeric mouse.

Specially designed transgenic animals are used to study gene regulation and effects of genes on the normal functions of the human body and its development. To study the role of insulin in humans, genes from a rabbit or a mouse are introduced into another mouse, which then gives birth to transgenic animals having the altered gene for insulin. Then, the biological effects of the newly introduced gene are studied to obtain information about the role of insulin in the human body.

Transgenic animals are also used for understanding how genes contribute to the development of a disease and thereby help in treatments for diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s disease. Transgenic animals are used to produce expensive biological products such as alpha one antitrypsin used for the treatment of emphysema, at a cheaper rate. Even attempts for treatment of genetic disorders such as phenylketonuria or PKU and cystic fibrosis were made.

The first transgenic cow, Rosie, produced human protein enriched milk containing 2.4 grams of human protein in every litre. This milk contained the human gene alpha-lactalbumin which made it a more nutritionally balanced product than natural cow milk. The transgenic animals are also being used to test the safety of vaccines before using on humans and to test and study the toxicity of chemicals. The creation of transgenic animals has even reduced the overall use of laboratory animals.

Summary

The growth in the field of molecular biology and biotechnology is due to the intensive research using transgenic animals. However, there are many ethical issues regarding the use of transgenic animals, because, the genome of transgenic animals is deliberately modified. The first transgenic animals called chimeric mice were created by combining two cells taken from two different embryos of different strains. This gave rise to a single embryo, which was implanted into a surrogate mother to give birth to a chimeric mouse.

Specially designed transgenic animals are used to study gene regulation and effects of genes on the normal functions of the human body and its development. To study the role of insulin in humans, genes from a rabbit or a mouse are introduced into another mouse, which then gives birth to transgenic animals having the altered gene for insulin. Then, the biological effects of the newly introduced gene are studied to obtain information about the role of insulin in the human body.

Transgenic animals are also used for understanding how genes contribute to the development of a disease and thereby help in treatments for diseases such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer’s disease. Transgenic animals are used to produce expensive biological products such as alpha one antitrypsin used for the treatment of emphysema, at a cheaper rate. Even attempts for treatment of genetic disorders such as phenylketonuria or PKU and cystic fibrosis were made.

The first transgenic cow, Rosie, produced human protein enriched milk containing 2.4 grams of human protein in every litre. This milk contained the human gene alpha-lactalbumin which made it a more nutritionally balanced product than natural cow milk. The transgenic animals are also being used to test the safety of vaccines before using on humans and to test and study the toxicity of chemicals. The creation of transgenic animals has even reduced the overall use of laboratory animals.


Knockout Mice: What do they teach us?

If the replacement gene (A* in the diagram) is nonfunctional (a "null" allele), mating of the heterozygous transgenic mice will produce a strain of "knockout mice" homozygous for the nonfunctional gene (both copies of the gene at that locus have been "knocked out").

  • Knockout mice are often surprisingly unaffected by their deficiency. Many genes turn out not to be indispensable. The mouse genome appears to have sufficient redundancy to compensate for a single missing pair of alleles.
  • Most genes are pleiotropic. They are expressed in different tissues in different ways and at different times in development.

The Zebrafish: Genetics, Genomics and Informatics

Gembu Abe , . Koichi Kawakami , in Methods in Cell Biology , 2011

D Discussion

Transgenesis using the Tol2 transposon system is highly efficient. 50–70% of fish injected with the Tol2 system at the one-cell stage and grown up to the adulthood are germline-transmitting founder fish that can transmit the transgene to their offspring. In addition, Tol2-mediated transgenesis has the following merits. First, transgenic fish carrying a single copy transgene integration can easily be created, whereas transgenic fish constructed by the plasmid DNA injection method often carry transgene concatemers at a single locus whose expression is silenced ( Stuart et al., 1988 ). Second, end-to-end integration of the transgene is guaranteed. Third, the transposon insertion does not cause gross rearrangement of the integration locus. Since the integration site is clean, it can be analyzed by PCR-based methods such as inverse PCR ( Kawakami et al., 2000, 2004 ), and adaptor-ligation PCR ( Kotani et al., 2006 Urasaki et al., 2006 ).


Class 12 Biology Chapter 12: Transgenic Animals

Animals whose genomes have been modified are known as Transgenic Animals. A foreign gene is inserted into the original genome of the animal to change its DNA. This method is used to improve the genetic traits of the targeted animal. The first ever transgenic animal was chimeric Mice, they were created by combining two cells taken from two different embryos of different strains. 

In the starting phases, genetic improvement was done by selective breeding methods. In which, the animals having required genetic qualities were mated for producing Individuals with Improved genetic characteristics. This method was quite successful, but was later replaced by recombinant DNA technology due to higher time consumption and higher expenses.

Define Transgenic Animals

The process of production of transgenic animals is known as Transgenesis. In which a foreign gene with desired qualities is introduced into the genetics of the targeted animal. The foreign gene introduced is known as the Transgene, and the Animal whose genome is changed is known as Transgenic. The genes are then passed further on to the next generations. 

Specifically designed transgenic animals are used to study the effects of genes on normal functioning of the human body & its development. They are also used to understand how genes contribute to the development of a disease and then help in treatments of various diseases. Transgenic animals are also used to produce expensive biological products at cheaper rates.Transgenic animals are genetically modified, that is why they are also known as Genetically Modified Animals (Organisms). 

ਏigure: The process of production of Transgenic animals

Methods For Producing Transgenic Animals

The Transgenic Animals are created by the various methods explained below : 


Physical Transfection

In the Physical Transfection method, the Gene of desired characteristics is directly injected into the pronucleus of the Fertilised Ovum. This was the first method to become effective in Mammals and was applicable to a large variety of species. Some other methods of Physical Transfection are particle bombardment, ultrasound & electroporation.


Chemical Transfection

One of the Chemical processes of gene Transfection is Transformation. In this method, DNA of the targeted animal is taken up in presence of Calcium Phosphate. In this method, the DNA & calcium phosphate co-precipitates, which eases in DNA uptake. The mammalian cells have the ability to take up foreign DNA from the culture medium. 


Retrovirus-Mediated Gene Transfer 

In order to enhance the chances of expression, the genes are conveyed by means of a vector. As retroviruses are capable of infecting the host cell, they are used as vectors so that they can transfect the desired genes into the targeted animal&aposs genome.


Viral Vectors 

In this method, various viruses are used to transfect rDNA into the Animal cell. The viruses have the ability to infect the Host Cell, express well as well as replicate systematically. 


Bactofection 

Bactofection is the method by which genes of interest are transferred into the genome of the targeted animal with the help of bacteria.

Some Transgenic Animals

Some examples of Transgenic Animals are as follows,
Transgenic Mice

The process wherein by injecting DNA into oocytes or 1-2, the transgenic mice are developed is called Embryos taken from Female Mice. After injecting the DNA, the Embryo is implanted into the uterus of Receptive Females.

Figure: Transgenic mice.

Dolly Sheep

Dolly, the sheep was the very first mammal to be cloned with the help of an adult cell. In this process, the udder cells from a Finn Dorset white sheep of 6 years old were injected into an unfertilized egg from a Scottish Blackface ewe, whose nucleus was removed. With the help of electrical pulses, the cell was made to fuse. After fusion of the cell nucleus with the egg, the resulting embryo was cultured for the next 6-7 days. The embryo was then implanted into another Scottish Blackface Ewe, which was responsible for the birth of Transgenic Sheep, Dolly .

Some Applications of Transgenic Animals 

It is because the benefits are provided to the man that the Transgenic Animals are being created. Some of them are given below: 


Biological Products

Various biological products such as Medicines & Nutritional Supplements are obtained from transgenic animals. Research is still going on for manufacturing medicines in order to treat the diseases like hereditary emphysema and phenylketonuria. The first Transgenic Cow, produced milk which contained 2.4 gram human protein per litre. This milk can also be given to babies as a substitute of natural cow milk.

Normal Physiology and Development

Foreign Genes are introduced into Transgenic Animals, due to which the growth factor alteres. Which is why these animals are helpful for the study of Gene Regulation and their effects on the Functioning of the Body.


Study About Diseases

These animals are specially structured for the analysis of the role of genes in the development of certain diseases. Additionally, in order to come up with a cure for these diseases, the transgenic animals are used as model organisms.

For instance, there are various Transgenic models for diseases such as Alzheimer&aposs and Cancer.


Vaccine Safety

Before the vaccines are injected into humans, the transgenic animals are used as model organisms. This is for testing the safety of the vaccines.

Frequently Asked Questions

Que. Can the Transgenic Animals created in labs be released into the Ecosystem?

Ans. No , because if they are released into the Ecosystem they can spread to the natural population and cause an imbalance in the Ecosystem.

Que. How does a transgenic cow differ from a Normal/Natural cow?

Ans. Transgenic cows are the genetically modified (GM) cows. An extra gene or genes gets inserted into their DNA. That Extra Gene may come from the different species or from the same species .

Que. Are knockout mice also a Example of transgenic Animals?

Ans. Yes, they are also Genetically Modified Mouse in which an existing gene has been Inactivated by replacing it with an artificial piece of DNA.

Que. What was the unique Application of the milk obtained from the first transgenic cow ?

Ans. The First ever transgenic cow (Rosie) gave milk which had human alpha-lactalbumin. It was a more balanced product nutritionally for human babies than milk of a natural cow.

Ques. What are the Ethical Issues of Transgenic Animals ?

Ans. Genetic Engineering and selective breeding violate the animal rights as they involve manipulating animals .In this the animals are used for the Wellbeing of Humans.Rather than treating them being of value in themselves .

Que. What are the methods of creating transgenic animals?

Ans. The methods of creating transgenic animals are,

Physical transfection- In the Physical Transfection method, the Gene of desired characteristics is directly injected into the pronucleus of the Fertilised Ovum. This was the first method to become effective in Mammals and was applicable to a large variety of species. 

Chemical transfection- One of the Chemical processes of gene Transfection is Transformation. In this method, DNA of the targeted animal is taken up in presence of Calcium Phosphate. In this method, the DNA & calcium phosphate co-precipitates, which eases in DNA uptake. 

Retrovirus-mediated gene transfer- In order to enhance the chances of expression, the genes are conveyed by means of a vector. As retroviruses are capable of infecting the host cell, they are used as vectors so that they can transfect the desired genes into the targeted animal&aposs genome.

Viral vectors- In this method, various viruses are used to transfect rDNA into the Animal cell. The viruses have the ability to infect the Host Cell, express well as well as replicate systematically. 

Bactofection- Bactofection is the method by which genes of interest are transferred into the genome of the targeted animal with the help of bacteria.


Transgenes in genetically modified food are safe for human consumption

So, you’ve bought a new pair of jeans and you love them. You wear them every single day…you even sleep in them. They were made with transgenic cotton, so your skin, the largest organ in your body, is in constant contact with the transgenic fibers of the cotton. Should you be worried that you’ll absorb the bollworm toxin from the cotton through your skin and be poisoned by it? The answer is no, and here’s why:

  1. The cotton is dead plant tissue. Its cells are no longer expressing genes, including that toxic transgene.
  2. The transgene was toxic to bollworm larvae, not to mammals.

Okay, so the transgenic cotton you wear cannot harm you. But what about the transgenic food we eat, like tomatoes, soy, or corn? Most of the corn and soy grown in the US is transgenic. Why shouldn’t we worry about eating transgenic (versus non-transgenic) plants?

  1. As with cotton, the food plant DNA and the transgenes in it are in the plant cells, which are dead by the time they reach your table, much less enter your digestive system. They do not have the ability to express themselves, and the proteins those genes make are not harmful to mammals.
  2. Your body absorbs nutrients (vitamins, minerals) and sugars from food in the intestine. The plant matter itself, including the DNA, stays on the “outside” of your body, because the digestive system is basically just a long tube that runs through your body but never connects to the inside. It only has two openings, the mouth and anus, and both of those go to the outside of the body.

Transgenic Fly Virtual Lab

This interactive, modular lab explores the techniques used to make transgenic flies and demonstrates how these flies can be used to study gene expression.

Scientists use transgenic organisms, which contain DNA that scientists inserted in the organisms’ genomes, to research many biological processes. In this lab, students produce and conduct experiments with virtual versions of transgenic Drosophila fruit flies. Students first create transgenic flies that glow when a gene involved in circadian rhythms is activated. They then use these flies in three experiments to examine gene expression under different conditions and in different locations in the fruit fly’s body. Throughout this lab, students engage in key science practices, including evaluating a hypothesis, collecting data, and interpreting graphs.

The lab contains an interactive lab space, an informational notebook, and embedded quiz questions. It also includes supplementary resources, such as a glossary of scientific terms, images of equipment and tools, and a list of references.

The accompanying worksheet provides structure and guidance as students perform the tutorials, experiments, and quizzes in the lab.

Student Learning Targets
  • Describe how recombinant DNA technology is used to produce transgenic organisms.
  • Explain how transgenic organisms can be used to explore biological processes.

Explain how light production through a reporter gene is used as an external marker of internal molecular events.


Watch the video: 25. Θνησιγόνα γονίδια και Πολλαπλά αλληλόμορφα 5 5ο κεφ. - Βιολογία Γ λυκείου (June 2022).


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