In 1980, the advance in molecular biology and transgenic changed many perspectives in sciences and medicine toward gene therapy and enabled human genes to be sequenced and cloned. The scientists looked for the method in which proteins can be produced easily such as insulin - the protein deficient in diabetes. Scientists started by introducing the human genes to bacterial DNA. The bacteria that has been modified produces corresponding protein which then can be injected into people who don't produce it naturally.1
The application gene transfer techniques have furnished new insights in the development of biology and concepts underlying cells specific gene expression. The gene transfer allows the new production system development for pharmaceutically important proteins. The gene transfer techniques appear to be promising for improving disease resistance performance of the quality animal products through modification e.g. metabolic pathways and hormone status.2, 3
There are various definitions for the term transgenic or transgenic animal, it has been defined as the animal in which its genome has been modified. The genetic makeup of an organism that responsible for inherited characteristics. By other meaning transgenic animal is the animal that carries foreign gene which has been deliberately inserted into its genome by using recombinant DNA method.4
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Many transgenic animals have been produced such as mice, rabbits, rats, sheep, pigs and cows, although many ethical issues surround the transgenic. My writing focus on the application of transgenesis in medicine and the basic technology in which transgenic animal produced, evaluating how a disease model in transgenic mouse helped in treatment of various diseases such as Î²-thalassemia, cystic fibrosis etc.
1. Transgenic mice as a model for human disease
Transgenic mice have been used quite extensively as models for various hormone disease, transgenic models for human disease have become more refined and currently for pathological mechanisms study behind the disease rather than just providing a model of the disease. Transgenic mouse contains artificially additional genetic materials in every cell, which confers a gain of the function, e.g. the mouse may synthesis a new protein; however it may not function if the integrated DNA disturbs another gene. A transgenic mouse is a very useful model for studying mammalian or specifically human gene function and regulation because the analysis is basically carried out on the whole organism. Transgenic mice are used also as a model for human disease involved in misexpression or overexpression of particular protein.5, 6
More than 80% of mouse genes function the same as genes in human. Mice have also a short reproduction cycle; therefore, mice are an ideal model to study most of human disease. At present over 95% of transgenic animals are mice, that is why mice are widely used in biomedical research.6
Two methods of producing transgenic mice are widely used, these are:
By introducing or injecting the desired gene into the pronucleus of fertilized mouse egg
By transforming embryonic stem cells ( ES cells ) that grow in tissue culture with desired DNA.
2. Knock-out and knock-in technology
Knock-out and knock-in models are ways to target a mutation to specific gene locus. These methods are useful particularly if a single gene is shown to be the main cause of the disease. Knock-out mice carry a gene which has been inactivated, and that creates less expression and loss of function. Knock-out referred to the mice generated with specific genetic mutation Knock-in mice are produced by the inserting a transgene into an exact location where it is overexpressed. Over the years there are more than 3000 genes of mice have been knocked-out, most of these genes are related to diseases.7 In transgenic animals as models for human
Both knock-out and knock-in models are created in the same way, a specific mutation is inserted into the endogenous gene. Then it is transferred to the next generation by breeding. The use of ES cells is required in this technology, because ES cells can contribute to all cell lineages when they are injected into blastocytes, and they can be modified genetically and selected for the desire gene changes. Homologous recombination makes the mutation. This process is physically rearranges two strands of DNA for the exchange of genetic materials. In this way, many types of mutation can be introduced into an animal gene including point or null mutation and complex chromosomal rearrangements, such as translocation,3 deletion or inversion.8 many knock-out and knock-in mice have similar phenotype if not identical to human patients and therefore they are good models for human diseases. The production of knockout mouse is summarized in figure10.
3. The benefits of transgenic animals to human welfare can be classified into:
Always on Time
Marked to Standard
Agriculture a pplications
Scientists can make transgenic cows to produce more milk with less cholesterol or less lactose and farmers are using selective breeding to develop traits in animals in a short time. In addition scientists attempt to produce farm animals that are resistant to disease such as influenza.10, 11, 12, 13
Transplant organs (future hope) may come soon from transgenic animal. Pharmaceutical and nutritional supplements such as growth factors, insulin and blood anti-clotting factors may be obtained or already have been made from the milk of transgenic cows, goat or sheep.14, 15, 16
Pharmaceutical industry have produced enzymes that can speed up the industrial chemical reaction and produced toxicity-sensitive transgenic animals for chemical safety testing.17
4. Beta thalassemia-a human disease model in transgenic mouse:
In my writing I focused on a transgenic mouse model produced from lentiviral germline integration, to investigate and evaluate the gene therapy for Î²-thalassemia patients. Also how the suitable vector helped in mediating the generation of human Î²-globin in a Î²-thalassemia mouse and what are the consequences of the study in term of therapeutic effect in Î²-thalassemia.
Background: Thalassemia is congenital, quantitative defects in the synthesis of haemoglobin that is mostly found in the temperate regions of the world. Î²-thalassemia is an autosoml recessive single gene disorder caused by reduction or absence in Î²-globin chain production. In Î²-thalassemia the defect globin gene is due to decrease or no synthesis of Î²-globin chains that encodes the Î²-chain of adult haemoglobin (HbA). The Î²-globin chain defect leads to imbalance of Î² and Î±-globin chain, resulting in intracellular precipitation of excess Î±-globin chain and ineffective erythropoiesis. In most of the sever forms found in homozygotes or compound heterozygote, the anaemia is fatal in the years of life in the absent of any treatment. The majority of patients with Î²-thalassemia require lifelong transfusion therapy to suppress the massive erythropoiesis and to correct anaemia. However, transfusion therapy increase the burden of iron overload which must be treated aggressively to avoid sever or fatal complication. The only definitive cure for the disease is bone marrow transplantation when a related matched donor is available. However, sever complication may still arise after BMT. Globin gene therapy would be applicable to every patient with a reduced myeloablative regimen and could be preferable to BMT.but, globin gene transfer particularly in its stringent transcriptional requirement, transgene expression and differentiation stages have to be specific. 18, 19, 20
4.2 Molecular basis of Î²-thalassemia:
Î²-globin gene (HBB) is located on the short arm of chromosoms11. Normally, there are two Î²-gene on chromosom11 and two pairs of chromosomes. The single gene disorder in Î²-thalassemia resulting in either Î²Â°-thalassemia is produced which characterized by total absence of Î²-globin chain production or Î²+-thalassemia is produced which characterized by reduction of Î²-globin chain synthesis.21, 22
4.3 Classification of Î²-thalassemia:
According to the number of the genes mutated and clinical severity, Î²-thalassemia can be classified into three groups:
Î²-thalassemia minor: this is usually asymptomatic and it is a carrier type of Î²-thalassemia. Patients of this group have one mutated gene and one normal gene, their genotypes possibly are Î² /Î²Â° and Î²/ Î²+.
Î’-thalassemia intermedia: patients of this group have two abnormal genes. However, still some Î²-globin are produced and their possible genotypes are: Î²+/Î²Â° and Î²+ /Î²+.24
Î’-thalassemia major: It is known also Cooleys anemia, it is the most sever type of Î²-thalassemia because both Î²-globin genes are mutated, resulting in a complete absence or marked decrease of Î²-globin chain production. The patients suffering from Î²-thalassemia major have Î²Â° /Î²Â°, Î²+/ Î²+ and Î²Â° / Î²+ genotypes.24
4.4 Application of gene therapy for Î²-thalassemia using lentiviral in a mouse model.
The first alleviation of Î²-thalassemia symptoms in mouse model has been reported in 2000, after engraftment of the bone marrow cells stably transfused with lentiviral vector (TNS9) carrying large fragments of human Î²-globin gene. Long-term improvement of clinical symptoms of human Î²-thalassemia was reported using lentiviral vectors.25, 26
Substitution of C T at position IVS-2 nt 654 (Î² IVS-2-654 ) in human Î²-globin gene is the most common Î²-thalassemia alleles in the Chinese. Mutated patients with (Î² IVS-2-654 ) lead to spliced Î²-globin mRNA with approximately only 15% normal Î²-globin gene expression and it has been reported the generation of a human Î²-globin transgenic mouse in (Î² IVS-2-654 ) thalassemia model mediated by lentiviral vector to investigate the therapeutic effect on the common thalassemia syndrome.27, 28
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Human Î²-globin gene fragment was amplified by PCR from the human genomic DNA with specific primers which contained the restriction enzyme Xbal, Kpnl cutting sites-forward,5'-TGCTCTAGAGCTCCAGATAGCCATAGAAGAACC-3'-Xbal,
reverse-5'-GGGGTACCGCGAGCTTAGTGATACTTGT-3'-Kpnl, the cutting sites are highlighted. The amplified fragment is digested with Xbal and Kpnl, then cloned into the corresponding sites of LV. The most significant features of lentivirus as CMV enhancer substituted for the U3 region of the 5'LTR to maximize the expression of viral RNA genomes, and the deletion in U3 region of the 3'LTR to render the 5'LTR of the integrated previous.29
The LBG vector was contransfected with VSVG and R8.9 into the human embryonic kidney cells. Then the supernatant of the virus were collected after transfection at 72 hours and concentrated by low speed filtration using 0.2Âµm polyethersulfone filter,
followed by high speed centrifugation (26.000 rpm) at 4C for 90 mints. Next, the viral suspension was quantified by measuring the levels of gag protein in viral stocks, the final viral concentration of the viral preparation was approximately 2 Ã- 108 U/ML.8 The advantages of using retroviral vectors are:
Most of retroviral don't destroy their host cells
Easy to transfer genes in suitable target cells
High efficiency and a single copy integration without prestimulation with cytokines and can intact a fully nuclear membrane.
Abe to package full-length due to presence of strong RNA export element.
Can hold a large cargo (9-10kb) of genes
4.4.1 Generation of transgenic mice and the efficiency of lentiviral transgenesis by subzonal microinjection.
The Î² IVS-2-654 thalassemic male mice were mated superovulated type female, after treating erthropoietic cells taken from Î² IVS-2-654 thalassemic mice with oligonucleotides antisense that have been cultured for 14 days and reinjected in a wild type mouse Single embyos cells were collected and injected with viral particals of LBG by sub-zonal microinjection. Each embryo was injected with 10 U/ML. After that immediately implanted in the oviduct of the pseudo-pregnent wild type mice and permitted to develop full term and provide to founder transgenic mice(F0). The production of F1 & F2 generation was achieved respectively with wild type mice. 34.7% of live-born mice showed to be positive for LBG integration by PCR analysis, and 18.4% embryos developed to full term. The birth rate of transgene integration showed significantly high live-born pups when using subzonal microinjection method, whereas did not differ greatly from those generated by pronuclear injection.29
4.4.2 The consequences: Repair of splicing in erythroid cells in Î² IVS-2-654 thalassemia mouse, Improvement of RBC morphology and correction of hematologic indices.
After 15-17 days, blood sample was collected from the tail of the mouse and analysed for RNA by PCR and it is significantly observed the high level of correction of splicing in erythroid precursor. The repair of splicing occurs after approximately two weeks which thought to be the ideal time for oligonucleotides antisense to enter the nucleus, it seems that the neuclear compartment is the main site of antisense activity. The effectiveness of antisense in restoration of the correct splicing of IVS-2-654 mRNA in cultured erythropoietic cells from transgenic mouse and thalassemic patients suggesting the possible treatment of thalassemia.30
Moreover, Increased the survival rate of Î² IVS-2-654 mice that carry human Î²-globin transgene suggest according to mendeltion inheritance a 50% heterozygote of offspring between heterozygote Î² IVS-2-654 thalassemia and wild type mice and consequently suggesting a positive therapeutic effect of the human Î²-globin transgene. In addition the mice with stable expression of human Î²-globin showed significant improvement in red blood cell parameters e.g. the Hb increased from 10 g/dl to 11.7 g/dl and MCV from 47.2fl to 50.9 fl. Moreover, a dramatic reduction of reticulocytes from 14.71% to 11.29% was found, a marked decrease in red cells morphology ( anisocytosis, poikilocytosis and taget cells) have been noticed as well. 29
Significant levels of correction of splicing in erythroid cells in Î² IVS-2-654 thalassemia mouse was achieved by the treatment of oligonucleotides antisense, suggesting that the antisense should be applicable to studies on thalassemia on thalassemic mouse and possibly future treatment of thalassemia patients.31
On the other hand, it is believed that the generation of human Î²-globin transgenic mice by using lentiviral vector is useful and thought to be informative for pre-clinical assessment of the lentiviral mediated gene therapy in thalassemia. Subzonal microinjection method proved to be simple and more effective method for the generation of transgenic mice as it is showed a positive outcome and thus the therapeutic approach on common thalassemia.