This action might not be possible to undo. Are you sure you want to continue?
TERM PAPER – CONCEPTS IN BIOTECH
SUBMITTED TO: MR.HARSH
SUBMITTED BY:Amandeep Sharma B.Tech Biotech Sec:- B Roll No:- 19 Group :- G1.
Nothing concrete can be achieved without an optimal of inspiration and perspiration. And thanking people who have contributed to guide me is a little like saying thank you at the academic awards. But still I must dilute our feeling enough so as to fit them into framework of words and acknowledge .I would like to take a moment to thank those invaluable suggestion and support led to the completion of this Term Paper. I acknowledge the contribution of our teacher Mr. Harsh. Last but not least I am very grateful to all those who helped me in one or other way at every stage of my work.
TABLE OF CONTENTS 1-TRANSGENIC ANIMALS METHOD OF PRODUCING TRANSGENIC MICE 2-THE PRONUCLEUS METHOD TRANSFORM FERTILIZED EGGS 3-KNOCKOUT MICE 4-TISSUE SPECIFIC KNOCKOUT MICE 5-THE CRE/LoxP SYSTEM 6-TRANSGENIC SHEEPS AND GOATS 7-TRANSGENIC CHICKENS 8-TRANSGENIC PIGS
A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to a structural gene, 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 the eggs.
These animals should eventually prove to be valuable sources of proteins for human therapy. Transgenic mice have provided the tools for exploring many biological questions.
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:
growing in tissue culture with the desired DNA. Injecting the desired gene into the pronucleus of a fertilized mouse egg. 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 structural 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 pseudo pregnant 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 1:4 that will be homozygous for the transgene.
3. Implant the embryos in a pseudo pregnant foster mother and proceed as in Method 1. An 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 structural 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 gancyclovir.
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", 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. 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 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 imperfect.) 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 ribosome for efficient translation of the
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 have now 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; Synthesize several grams of protein in the "white" of their eggs.
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 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.
Applications of transgenic animals:
Transgenic produced animals can be of
cloned DNA into fertilized oocytes and cells from very early stage embryos
embryonic stem (ES) cells powerful route to genetic
modification of the germline
animals have been used for of studies investigating and gene expression
extended the applications of transgenic
REVIW OF LITERATURE:1.Genetically modified organism (GMO):As described by Holst-Jensen (2001) genetically modified organism (GMO) is a living organism, e.g. bacteria, plant, animal, whose genetic composition has been altered by means of gene technology. The genetic modification usually involves insertion of a piece of DNA and/or synthetic combination of several smaller pieces of DNA, into the genome of the organism to be modified. This process is called transformation. These DNA pieces are usually taken from other organisms such as bacteria or virus. A typical insert (gene construct) in a GMO is composed of three elements: 1) The promoter element functions as an on/off switch for reading of the inserted gene(s); 2)The gene(s) that has been inserted, which coding for a specific selected feature; 3) The terminator element functions as a stop signal for reading of the inserted gene(s). In addition, several other elements can be present in a gene construct, and their
function is usually to control and stabilize the
function of the gene, demonstrate the presence of the construct in the GMO or facilitate combination of the various elements of the construct. The area
planted with genetically modified (GM) crops, including soybeans and maize has increased worldwide in recent years. Between 1996 and 2002, it rose from 1.6 × 106 to more than 58 × 106 hectares (James, 2002) (Figure 2). Recent report released by James (2003) mentioned that worldwide plantings of biotech crops increased 12 percent in 2002. Between 5.5 million and 6 million farmers in 16 countries planted biotech seeds in 2002, up from 5 million farmers in 13 countries in 2001. By the same way biotech plantings will likely increase. Among the other interestingly findings in this report: • The area planted with biotech crops increased 35-fold between 1996 and 2002. • Four countries accounted for 99% of the global biotech acreage [Argentina(23%), Canada (6%), China (4%) and the United States (63%)]. The other 12countries accounted for the remaining 1% [Australia, Bulgaria] · The three new countries to begin planting biotech crops in 2002 were Colombia,Honduras and India. Columbia and Honduras conducted “pre-commercial” biotech plantings in anticipation of full commercial approval in 2003. · Four crops accounted for 99% of the global biotech plantings: Soybeans (63%), maize (19%), cotton (13%) and canola (5%). Biotech squash and papaya each accounted for less than 1%. This rapid increase has provoked an explosion of concern over the health and environmental impacts of these crops. Despite claims of safety and warnings against popular panic, public concern over GM crops has resulted in changes in their marketing, labelling, planting, and trade.
2. Actually present and future of genetically modified crops:-
Gaede (1997) and Flachowsky et al. (2002) classified edible crops developed by modern biotechnology from the nutritional point of view into two generations: Worldwide production of GM soybeans and maize 1996-2002 (James, 2002) - 1st Generation: Feed plants are characterized by changed tolerance or resistance to insects, herbicides, pesticides or other influencing factors with minor changes in nutrient content (e.g. Bt-maize, Pat-maize, Roundup Ready soybeans™, etc.). - 2nd Generation: Feeds are characterized by substantial changes in the content of valuable or undesirable major ingredients (e.g. protein, amino acids, fat, fatty acids, starch, sugars, lignin, etc.) or minor ingredients (e.g. vitamins, minerals, enzymes, antinutritive substances,etc.).The following overview (Matissek, 1998) summarized the objects of molecular biological modifications in plants with respect to food and feed production.The objects of previous as well as prospective research work allow to distinguish 4 groups: A) Direct increase in yield by creating high productive varieties of plants. B) Enhancement of productivity by elimination or reduction of disturbing biotic andabiotic influences and limitations, for instance: • introduction of herbicide resistance. • control of pest and diseases. • reduced losses due to abiotic stresses. C) Improvement of post harvest management and storage features. D) Improvement of food and feed qualities by: • direct improvement of nutritive value through modulation of nutrient synthesis. • elimination of unfavorable ingredients and increase contents of health benefit substances.
3. Insect resistant Bt-maize:Shah et al. (1995) estimated the worldwide annual costs of chemical control of maize plant insect (European corn borer insecticides) at 3-5 billion US $. In addition, great losses in maize plant yield are still caused by insect pests. To overcome this economical as well as ecological impact of chemical insecticides and to reduce the permanent danger of residue accumulation of insecticides, genes encoding for the insecticidal protein of Bacillus thuringensis (Bt) have been inserted into maize and other crop species by biotechnological methods to increase the resistant of the GM plants to insect pests (Ives, 1996). Bt (Bacillus thuringiensis) is a naturally occurring soil bacterium that is found worldwide. A unique feature of this bacterium is its production of crystal-like proteins (Cry proteins) that selectively kill specific groups of insect larvae (Bt-delta-endotoxins). Gasser and Fraley (1989) reported that all types of Cry proteins revealed no toxicity to beneficial insects, animals or human.
RESEARCH METHODOLOGY:Abstract:-- The notion of directly introducing new genes
or otherwise directly manipulating the genotype of an animal is conceptually straightforward and appealing because of the speed and precision with which phenotypic changes could be made. Thus, it is of little wonder that the imagination of many an animal scientist has been captivated by the success others have achieved by introducing foreign genes into mice. The private sector has embraced transgenic livestock technology resulting in the formation of two new industries. However, before transgenic farm animals become a common
component of the livestock production industry, a number of formidable hurdles must be overcome.
FUTURE PROSPECTIVE :-
Male infertility has been considered a major contributory factor to infertility. The causes of spermatogenetic failure found in most cases of male infertility remain largely idiopathic. Unfortunately, there is no effective treatment to improve spermatogenesis for idiopathic male infertility patients. In tracytoplasmic sperm injection (ICSI) is the current treatment of choice for severe male infertility and has brought the joy of childbearing to couples for whom it was previously impossible; however, several problems exist with this treatment. In addition, if there are no spermatozoa in the testis of these patients, they do not have paternity potential even if ICSI is conducted. Ultimately, fertilization is better in vivo than in vitro. Recently, on the other hand, gene transfer to sperm and testis has been developed to find more effective and simple methods to obtain transgenic animals. This technique has the potential to be the most useful approach for the future treatment of male infertility. In this review, we will give an overview of the recent advanced technique of gene transfer to sperm and testis, and discuss the future prospects of gene therapy for the treatment of male infertility. In conclusion, although more investigations on the mechanism of spermatogenesis and male infertility and the establishment of techniques for more efficient and safer gene transfer to the sperm and testis will be needed, gene therapy will enable a revolutionary advance for reproductive treatment and
provide great benefit for patients with male infertility in the future.
WEB LINKS www.google.com www.biospectrum.com
BOOKS Microbiology by Prescott. Concepts in Biotech By B.D.Singh. Basics of Animal Tissue Culture By Butler.