Inheritance And Gene Therapy In Cystic Fibrosis Biology Essay


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 [1]. 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 someone 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 [2].

2.0 The causes of cystic fibrosis

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Cystic fibrosis is caused by mutations in a gene responsible for production of a protein responsible for the movement of chloride ion during the cell membrane. This protein is known 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 absent of an amino acid in the (CFTR) protein. Mutations occur at position delta508 of the protein chain [3,4].

3.0 Inheriting copies of the allele in cystic fibrosis

A person have 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 every parent has a normal (CFTR) gene and a faulty (CFTR) gene, every child has a 25 % probability of inheriting two normal genes; a 50 percent chance of inheriting single normal gene and single faulty gene; and a twenty five percent chance of inheriting two defective genes [5].

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.

Other complications

Fingers and toes: rounding of the tips(clubbing).

Low bone density (osteoporosis) [6].

5.0 Diagnosis

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.

6.0 Treatment

Cystic fibrosis patients must be treated with assist and recommendation from healthcare professionals group 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 assist the person breathes easily. Antibiotics are taken to destroy and kill 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 enzymes have be taken which they assist the digestive system break down food so, that it can be digested and absorbed smoothly. Fat-soluble vitamin supplements (A, D, E and K) are taken to assist out restore lost vitamins and to avoid deficiencies. Insulin patients who have diabetes as a consequence of their cystic fibrosis will require taking insulin and controlling their diet to maintain blood glucose levels [7].

In addition hypertonic saline is the primary airway-rehydrating agent which is used in the therapy of cystic fibrosis disease. Inhaled mannitol may become an alternative to hypertonic saline since it is faster and easier to administer. Both agents have a single mechanism of action that bypasses the basic CFTR gene defect [8].

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Physiotherapy is an airway process 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. Cystic fibrosis mutation is concerned in the deletion of three of the base pairs (Phe) in the gene is known as deltaF508 [9,10].

Normal (CFTR)


Aminoacid Asn Ile Ile Phe Gly Vel Ser



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 a correct gene in affected tissues. This process known as gene therapy. So, the gene therapies in cystic fibrosis involve inhaling a spray that delivers usual DNA to the lungs.

The aim is to replace the malfunctioning cystic fibrosis gene in the lungs to treat cystic fibrosis disease or sluggish the progression. By vectors deliver a functional copy of the defective gene-in this disease, CFTR-either to cells during the body or to exact affected tissues such as the lungs. Generally vectors are normally derived from viruses that are able to be infecting the target cell. When the new CFTR gene has entered to the cell, the cell's biochemical machinery have to recognize it and apply it as a model used for the production of efficient protein [11].

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.

8.0 Transgenetic

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 throughout many perspectives in agriculture, food, medicine, industry and science. So, transgenic organism can be in animals, plants, and bacteria. Transgenic animals are helpful 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 diverse embryo of diverse 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. Also mice have a small 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

Production of transgenic mice was success by using pronuclear microinjection which has 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 badly arrange DNA can be toxic to the mouse eggs and contaminants can block the microinjection needle that usually has 0.5 microns diameter at the tip [14].

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1- DNA (douple-starnd) components of the transgene are shared enzymatically to give up a transgene expression cassette (restriction enzyme and ligase).

2- Transgene cassettes are interested in plasmid vectors and cloned.

3- Transgene-bearing plasmids are transfected in cultured eukaryotic cells to estimate expression of the transgene.

4- Plasmid-free transgene fragments are introduce directly in embryonic pronuclei.

5- Manipulated embryos are positioned in the reproductive tract of a pseudopregnant receiver.

6- Genomic DNA of live-born mice is analyzed for the attendance of the transgene DNA sequence.

This involves option 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 altered 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 allow the expression of gene completely new to animal. The species manipulated fertilized ovum is now transfer and interested in 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 [15].

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 techniques are useful mostly if a single gene is shown to be the main cause of the disease. Knock-out mice hold a gene which has been inactivated, and that creates less expression and failure 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 similar way; a exact mutation is inserted into the endogenous gene. Then it is transferred to the next generation by breeding. The use of embryonic stem cells is required in this technology, because embryonic stem cells can give to all cell lineages when they are injected into blastocytes, and they can be modified genetically and sure for the desire gene changes. Homologous recombination makes the mutation. This procedure is physically reorder DNA double strand for the replace of genetic information's. So, many types of mutation be able to introduced into an animal gene include point or null mutation and complex chromosomal rearrangement, such as translocation, three deletions or inversion. A lot of knock-out and knock-in mice have alike phenotype if not equal 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 method 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 phase of progress. The product 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 studies of genetic organize of developmental process and works particularly well in mice with this method and precisely target a defined mutation in the gene by homologous recombination [17].

8.3 Retrovirus-mediated gene transfer method

Gene addition inserts a functioning copy of a nonfunctional origin gene. Viral based gene addition involve the "domestication" of viral genomes as vectors. A retrovirus is viruses that take its genetic information in the form of RNA instead than DNA. Retroviruses used as vectors to transfer genetic information into the host cell, producing in a chimera, an organism contain of tissues or parts of diverse genetic material finally, 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 [18].

Retroviral genomes contain three genes significant in the infection cycle. First of the retroviral genes gag, encodes structural proteins. The second pol, encodes a variety of enzymes, contain reverse transcriptase. Finally, env 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 now faces application of the gene addition therapy that is the failure to identify the insertion point of the recombinant genes

8.4 DNA introduce by the Cre/loxP System

A number of site-specific recombination systems from yeast and bacteriophages have been considered and are promising tools for genome engineering ( Kilby., et al, 1993).

Cre/ loxP is one of the bacteriophages that infect E.coli, called P1, produces an enzyme -designated Cre - that cuts its DNA into lengths proper for packaging into fresh virus particle.

Cre cuts the viral DNA anywhere it meets a pair of sequences selected loxP. The entire DNA between the two loxP sites is removed and the remain DNA ligated together again, this enzymes called recombinase [20].

Mice can be made transgenic for

Gene encoding Cre close to a promoter that will be activated just when it is bound by the similar transcription factors that change on the other genes necessary for the single functions of that form of cell.

A target gene the one which role is to be studied, by loxP sequences.

9.0 Benefits of transgenic animals to human welfare can be classified:

Agriculture applications

Scientists can make transgenic cows to produce extra milk with less cholesterol or a reduced amount of lactose and farmers are using selective breeding to develop character in animals in a short period. In addition scientists attempt to produce farm animals that are resistant to disease such as influenza [21].

Medical applications

Patients die every year for need of a replacement heart, liver, or kidney. Transplant organs (xenotransplantation) may approach 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, sheep and goat [14]. In addition human gene therapies live in adding up a normal duplicate of a gene (transgene) to the genome of a person delivery faulty copy of the gene. The possible for treatments for the five thousands named genetic diseases is enormous and transgenic animals might play a function for patient treatment [22].

Industrial applications

Pharmaceutical industry has produced enzymes that can speed up the industrial chemical reaction and produced toxicity-sensitive transgenic animals for chemical safety testing [23].

10.0 Consequences

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. These studies make the viability of gene therapy for the pulmonary aspects of cystic fibrosis in human [24,25].

11.0 Conclusion

Cystic fibrosis is a threatening inherited disease of exocrine glands, affecting mostly the pancreas, sweat glands and respiratory system. Usually beginning in infancy by chronic respiratory infections, pancreatic deficiency and digestive problems. The gene for a chloride channel (CFTR) is affected, whose function is then limited and leads to mucilaginization, for the most part in the respiratory tract. Secondary infections with Pseudomonas aeruginosa often consequence from that.

Interestingly, the making of transgenic animals has assist in potential a change in the use of laboratory animals from order the big species such as dogs to small species such as mice and has reduction the number of animals used in such carrying out tests, particularly in the progress of disease models. So, this is positively a good turn of actions since transgenic technology hold huge potential in many fields, counting in medicine, industry and agriculture.


Also, transgenic animals are used to increase our understand of how genes important to the developmental of disease. The animal is genetically engineered to produce the disease symptoms so, new and effective treatment can be studied.

Finally, gene therapy, in which normal genes are delivered directly to the airways, holds great promise in future for cystic fibrosis treatment.