This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.
Cystic fibrosis may be defined as a lethal inherited disease of the exocrine glands that affect internal organs especially respiratory and digestive system in children and young adult, causing them to become blocked with thick sticky mucus leading hard in breathing and digestion food, also affects sweat gland and male fertility . Figure1. Show symptom and sign of cystic fibrosis.
It is a recessive disorder inherited by an offspring from each of the parents carrying a mutated gene. That means a person gets cystic fibrosis only when he or she inherits two copies of mutated gene, one from each parents. It cause gradual disability in the patient, and finally leads to death. It is a common among Caucasian population and affects over 8,500 people in the United Kingdom, and rarer in people of African or Asian origin. Cystic fibrosis patients are not given a chance to live more than 38 years, however with improvement in treatment, they can now live longer .
Cystic fibrosis is caused by mutations in a gene responsible for production of a protein responsible for the movement of chloride ion through the cell membrane. This protein is called cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic-AMP control chloride channel and changed in airway epithelial from cystic fibrosis patients.
Mutation or malfunction of this protein causes the deletion of the base pair in the gene which in turn cause loss of an amino acid in the (CFTR) protein. The mutations occur at position delta508 of the protein chain [3,4].
3.0 Inheriting copies of the allele in cystic fibrosis
A person gets cystic fibrosis when inherits two mutated (CFTR) genes that is one from each parent. The child gets cystic fibrosis when he or she receives one of the chromosomes 7 from father and one from mother. If each parent has a normal (CFTR) gene and a faulty (CFTR) gene, each child has a 25 % chance of inheriting two normal genes; a 50 percent chance of inheriting one normal gene and one faulty gene; and a 25 percent chance of inheriting two faulty genes .
Figure 2. Diagram shows how CFTR genes are inherited in cystic fibrosis adopted from (Zielenski J. 2000).
4.0 The manifestations in cystic fibrosis
Many organs affecting in cystic fibrosis disease:
Sinuses : cause sinusitis (infection)
Lungs : cause thick sticky mucus build up, bacterial infection and block airways.
Skin : sweat glands salty produce salty sweat.
Liver : leading blocked biliary duct.
Pancreas: blocked pancreatic ducts.
Intestines: cannot fully absorb nutrients.
Reproductive system: leads to blockage of sperm duct in a male, causing infertility.
Fingers and toes: rounding of the tips (clubbing).
Low bone density (osteoporosis) .
Figure 3. show sign of clubbing finger. Adopted from (James H. (2006).Â
A blood test is available to diagnosis cystic fibrosis disease. Example:
1- Amniocentesis for genetic testing can be done through pregnancy.
2- Immunoreactive trypsinogen (IRT) test is a model newborn screening test for cystic fibrosis.
3- Sweat Test.
4- Liver function test.
5- Chest X-ray or CT scan.
6- Stool fat test.
7- Sputum culture.
Cystic fibrosis patients should be treated with assist and advice from a team of healthcare professionals at a cystic fibrosis centre. Various treatments exit for the symptoms and the complications of cystic fibrosis. The main goal is to prevent infection, decrease the amount and thickness of secretion in the lungs. Improvement of airflow and maintain sufficient calories and nutrition assist the patient. However, there are many treatments available:
In lungs and airways Bronchodilator drugs are inhaled to help the person breathe easily. Antibiotics are taken to fight infections in the lungs Staphylococcus aureus, Burkholderia cepacia and Pseudomonas aeruginosa are usually found. DNase is an enzyme, generally inhaled, so it is easier to cough. Whereas in Digestive system Pancreatic enzymesÂ should be taken which they assist the digestive system break down food so that it can be digested and absorbed easily. Fat-soluble vitamin supplementsÂ (A, D, E and K) are taken to help restore lost vitaminsÂ and to avoid deficiencies. Insulin patients whoÂ have diabetes as a result of their cystic fibrosis will need to take insulin and control their diet to maintain blood glucose levels .
In addition hypertonic saline is the primary airway-rehydrating agentÂ used in the treatment of CF. Inhaled mannitol may become anÂ alternative to hypertonic saline since it is faster and easierÂ to administer.Â Both agents have a single mechanism of action that bypassesÂ the basic CFTR gene defect .
Physiotherapy is an airway technique clearance is used to clear the air way from mucus secretion and lungs transplantation become more successful to improve the cystic fibrosis condition.
7.0 Gene therapy in cystic fibrosis
The single gene mutation is a cystic fibrosis transmembrane regulator (CFTR), is located 7q31.2, 250,000bp long, have 27 exons and protein has 1,480 aminoacids. The mutation involved in cystic fibrosis causes the deletion of three of the basepairs (Phe) in the gene is known as deltaF508 [9,10].
Nucleotide AAA ATC ATC TTT GGT GTT TCC
Aminoacid Asn Ile Ile Phe Gly Vel Ser
Nucleotide AAA ATC ATC GGT GTT TCC
Aminoacid Asn Ile Ile Gly Vel Ser
Cystic fibrosis finally might be cure if effective and safe found to replace the mutation in CFTR gene with an intact gene in affected tissues. This process known as gene therapy. So, the gene therapy in cystic fibrosis involves inhaling a spray that delivers normal DNA to the lungs.
The aim is to replace the defective cystic fibrosis gene in the lungs to cure cystic fibrosis disease or slow the progression. By vectors deliver a functional copy of the defective gene-in this disease, CFTR-either to cells throughout the body or to specific affected tissues such as the lungs. These vectors most normally are derived from viruses that can infect the target cell. Once the new CFTR gene has entered the cell, the cell's biochemical machinery must recognize it and apply it as a template for the production of functional protein .
7.1 Role (CFTR) protein
Proteins moves chloride ions out of an epithelial cell to the covering mucus, whereas in cystic fibrosis the chloride ions not movement and cause thick sticky mucus.
Transgenetic is the transfer of genetic information (DNA) that is not usually present into the genome of a species and study the consequences from addition that genetic material into an organism. Transgenetic and Biotechnology has changed the world during many perspectives in agriculture, food, medicine, industry and science.
So, transgenic organism can be in animals, plants, and bacteria. Transgenic animals are useful as disease models and for human welfare.
There are might be a various definitions for the term transgenic or transgenic animal, transgenic animals are animals that are genetically modified by modification of the genome. It involves introducing a foreign DNA into the animal genome via a recombinant DNA technology. This involve combination of cells of two different embryo of different strain at an early stage of development to form a single embryo that build up into chimeric adult exhibiting type of each strain. For the ease of experiment that is small size, lower cost, rapid reproduction rate, similarity to human, genetic diversity and time. Mice model have been selected for use in the field of transgenic in cystic fibrosis disease. A transgenic mouse is a very useful model for studying mammalian or specifically human gene function and regulation for the reason that the analysis is basically approved out on the whole organism. More than 80% of mouse genes have the same function as genes in human. Mice have also a short reproduction cycle; therefore, mice are an ideal model to study most of human disease. At this time over 95% of transgenic animals are mice that are why mice are widely used in biomedical research, and used as a model for human disease concerned in misexpression or overexpression of particular protein [ 12,13].
Transgenic mice can be manufactured by:
1- Pronuclear Micro-injection.
2- Embryonic Stem cell -mediated gene transfer.
3- Retrovirus-mediated gene transfer.
8.1 Pronuclear micro injection method
The first successful production of transgenic mice using pronuclear microinjection was reported in 1980 (Gordon et al., 1980).
Transgene DNA Preparation
Transgene DNA is a genetically engineered in the laboratory to give the animal positive qualities and traits which can help in a better survival. It is an important steps in making transgenic mice, because poorly prepared DNA can be toxic to the mouse eggs and contaminants can block the microinjection needle that usually has an inside diameter of 0.5 microns at the tip .
1- Double-stranded DNA components of the transgene are shared enzymatically to give up a transgene expression cassette (restriction enzyme and ligase).
Figure 5. Schematic diagram of the double-stranded DNA regions of the transgene expression cassette.
2- Transgene cassettes are inserted into plasmid vectors and cloned.
3- Transgene-bearing plasmids are transfected into cultured eukaryotic cells to estimate expression of the transgene.
4- Plasmid-free transgene fragments are introduce directly in embryonic pronuclei.
5- Manipulated embryos are located in the reproductive tract of a pseudopregnant recipient.
6- Genomic DNA of live-born mice is analyzed for the presence of the transgene DNA sequence.
Figure 6. Sequence of events in the generation of a transgenic animal by pronuclear microinjection.
This involves choice a needed gene from either single or a combination of gene (the chosen gene is known as a gene construct) from a new member of the same species or different species into the pronucleus of a fertilized ovum.
The introduced gene may insert or not meet if it does not it can be repeated until if inserts and permit the expression of gene completely new to animal. The species manipulated fertilized ovum is now transferred into the oviduct of a recipient females or foster mother ( to be mother) that has previously been induced to act as a recipient female or foster mother by mating with vasectomised male. This method can be applied to a variety of species .
8.2 Embryonic Stem cell -mediated gene transfer method
Knock-out and knock-in models are methods 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. 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 give to all cell lineages when they are injected into blastocytes, and they can be modified genetically and certain for the desire gene changes. Homologous recombination makes the mutation. This procedure is physically rearranges two strands of DNA for the exchange of genetic materials. So, many types of mutation can be introduced into an animal gene including point or null mutation and complex chromosomal rearrangements, such as translocation, three deletions or inversion. 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 [15,16].
Embryonic stem cells (ES cells) may be defined as a pluripotent stem cell which derived from the inner mass cell from the blastocyst. This technique requires a prior insertion of the required DNA sequence by homologous recombination into an invitro culture of the embryonic stem cells (ES).
The cells are then introduced into an embryo of the blastocyst stage of development. The result is a chimeric animal. Embryonic stem cell -mediated gene transfer is the process of choice for gene inactivation and this process known as a knock-out method. It is important for the study of genetic control of developmental processes and works particularly well in mice with this method and precisely target a defined mutation in the gene via homologous recombination .
Figure 7. Two methods of making transgenic mice
8.3 Retrovirus-mediated gene transfer method
Gene addition inserts a functioning copy of a nonfunctional origin gene.Â Viral based gene addition involves the "domestication" of viral genomes as vectors.Â A retrovirus is viruses that carry its genetic information in the form of RNA rather than DNA. Retroviruses used as vectors to transfer genetic information into the host cell, resulting in aÂ chimera, an organism consisting of tissues or parts of diverse genetic constitution then, chimeras are inbred for as many as twenty generations until homozygous transgenic offspring are born. Transmission of the transgenic is likely only if the retrovirus integrates into a few of the germ cells .
Retroviral genomesÂ contain three genes significant in the infection cycle. First of the retroviral genes,Â gag, encodes structural proteins. The second,Â pol, encodes various enzymes, include reverse transcriptase. Finally,Â envÂ encodes the envelope glycoprotein. Long Terminal Repeat (LTR) regulatory sequences, start at the insertion points of the genome, are important for integration of the virus interested in the host genome. The most disadvantages currently faces application of the gene addition therapy that is the failure to specify the insertion point of the recombinant genes Figure 8. Show Viral Vectors for gene addition .
Figure 8. Viral Vectors for gene addition Adapted from (Walters & Palmer, 1997).
8.4 DNA introduce by the Cre/loxPÂ System
A number of site-specific recombination systems from bacteriophages and yeasts have been characterized and are promising tools for genome engineering ( Kilby., et al, 1993).
Cre/ loxP is one of theÂ bacteriophagesÂ that infectÂ E.coli, called P1, produces an enzyme -designated Cre - that cuts its DNA into lengths proper for packaging into fresh virus particles.
Cre cuts the viral DNA anywhere it encounter a pair of sequences selectedÂ loxP. All the DNA between the twoÂ loxPÂ sites is removed and the remain DNA ligated together again, this enzymes called recombinase .
Mice can be made transgenic for
Gene encoding Cre attached to aÂ promoter that will be activated only when it is bound by the similar transcription factors that turn on the other genes required for the single functions of that type of cell.
A target gene the one whose function is to be studied, byÂ loxPÂ sequences.
9.0 Benefits of transgenic animals to human welfare can be classified:
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 .
Patients die every year for lack of a replacement heart, liver, or kidney. Transplant organs (xenotransplantation) 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 . In addition human gene therapies involve adding a normal copy of a gene (transgene) to the genome of a person carrying faulty copies of the gene. The possible for treatments for the 5,000 named genetic diseases is enormous and transgenic animals could play a role for patient treatment .
Pharmaceutical industry have produced enzymes that can speed up the industrial chemical reaction and produced toxicity-sensitive transgenic animals for chemical safety testing .
Transgenic mice with defect CFTR gene are appropriate for testing gene therapy protocols. By use of liposomes to deliver a CFTR expression plasmid to epithelial of the airway and to alveoli in the lung, due to the correction of the ion conductance defects found in the trachea of transgenic (cf/cf) mice. This study illustrates the viability of gene therapy for the pulmonary aspects of cystic fibrosis in human [24,25].
Cystic fibrosis an inherited disease of exocrine glands, affecting most characteristically the pancreas, respiratory system, and sweat glands, usually beginning in infancy and typified by chronic respiratory infections, pancreatic insufficiency and digestive problems. The gene for a chloride channel (CFTR) is affected, whose function is then limited and leads to mucilaginization, particularly in the respiratory tract. Secondary infections with Pseudomonas aeruginosa frequently result from that.
Interestingly, the making of transgenic animals has resulted in a shift in the use of laboratory animals from use of higher-order species such as dogs to lower-order species such as mice and has reduction the number of animals used in such experimentation, particularly in the development of disease models. This is positively a good turn of events since transgenic technology hold great potential in many fields, including agriculture, medicine, and industry.
In addition transgenic animals are used to increase our understand of how genes contribute to the developmental of disease. The animal is genetically engineered to produce the disease symptoms so, new and effective treatment can be studied.
Gene therapy, in which normal genes are delivered directly to the airways, holds great promise in future for cystic fibrosis treatment.