Molecular Biotechnology: Principles and Applications - Glick, B., & Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Transgenic Animals
Transgenic Mice: Applications

Transgenic mice can serve as model systems for studying human diseases and test systems for investigating the potential synthesis of medically relevant products. Whole-animal models allow researchers to simulate both the onset and progression of pathologies. However, although mice belong to the Class Mammalia, they are not human; consequently, data obtained from transgenic models cannot always be directly extrapolated to humans regarding clinical aspects. Nevertheless, in certain cases, they help elucidate key aspects of the Etiology of complex diseases. Taking this into account, scientists have developed murine models for various human Genetic Disorders, such as Alzheimer's disease, Arthritis, muscular dystrophy, tumorigenesis, Hypertension, neurodegenerative disorders, endocrine dysfunction, cardiovascular diseases, and many others.

Alzheimer's disease is a degenerative process leading to the loss of Cells in various Regions of the Brain. The earliest manifestation is memory impairment. As the condition progresses, it is accompanied by a loss of Abstract thinking capabilities, personality changes, speech disorders, and a decline in physical status. This pathology is observed in 1% of individuals aged 60 to 65 and in 30% of those over 80. Pathomorphological examination reveals neurofibrillary tangles within neuron Cell bodies and dense aggregates known as senile plaques near synaptic terminals (Fig. 19.10). In addition, conglomerates called amyloid plaques are found in the cerebral Blood Vessels. The primary component of both senile and amyloid plaques is the Aß protein (amyloid ß, ß-protein, ß-amyloid protein, ß/A4) with a molecular mass of 4 kDa. There are Aß variants with varying numbers of amino acid residues, such as Aß40 and Aß42. All of them are generated through the proteolytic Cleavage of the amyloid precursor protein (APP). The exact triggers for Aß accumulation remain unknown. Members of certain families with a high incidence of Alzheimer's disease carry Mutations in the APP Gene, suggesting that this gene plays a role in the onset of the disorder. Unfortunately, tracking the initiation and progression of Alzheimer's disease in detail within humans is not feasible, making an appropriate animal model an invaluable asset.

Fig. 19.10. Schematic representation of a neuron from the human Cerebral Cortex, indicating several histological features characteristic of Alzheimer's disease. Senile plaques containing amyloid aggregates and cellular debris form near the synapses. Neurofibrils, comprising aggregates of cytoskeletal and other Proteins, accumulate within the neuronal cell body. Other changes not shown here also occur.

Numerous transgenic mice carrying either the full-length APP gene or a portion of it under the control of a neuron-specific promoter have been generated. However, the majority of these animals showed no evidence of amyloid plaque formation, neurofibrillary tangles, neuronal cell death, or behavioral abnormalities. In contrast, animals carrying a transgene encoding the C-terminal 100 Amino Acids of APP—which includes the Aß protein region—exhibited Nervous Tissue degeneration analogous to that seen in Alzheimer's disease.

More adequate animal models for studying Alzheimer's disease were developed using transgenes containing APP Gene Mutations characteristic of certain families with a high prevalence of early-onset Alzheimer's disease (<50 years). In one group of such families, a phenylalanine is substituted for valine at position 717 of APP (APP-717), while in another group, Lysine and Methionine at positions 670 and 671 of APP (APP-670/671) are replaced by asparagine and leucine, respectively.

The transgene carrying the APP-717 mutation was constructed from APP cDNA by inserting modified introns between exons 6 and 7, 7 and 8, and 8 and 9. These introns were introduced because experimental data demonstrated that transgenes containing introns are transcribed more efficiently than intronless ones. The APP cDNA–intron construct was placed under the control of the platelet-derived growth factor ß-chain gene promoter, which is expressed in brain tissues (Fig. 19.11). This entire construct was designated as the PDAPP minigene. In Aging transgenic mice (older than 6 months) carrying approximately 40 copies of PDAPP, amyloid plaques formed, accompanied by neuronal death and memory deficits. The APP-670/671 construct, driven by a neuron-specific promoter, induced Alzheimer's-like symptoms in transgenic mice, including the overproduction of Aß42. Interestingly, neurofibrillary tangles were detected neither in aging mice carrying the PDAPP minigene nor in APP-670/671 transgenic mice; these structures possibly arise in humans as a secondary consequence of Aß42 overproduction. Three other genes are involved in The Development of human Alzheimer's disease: ApoE4, presenilin 1 (PS1), and presenilin 2 (PS2) genes. The presence of the ApoE4 allele of the ApoE locus, which is responsible for lipid transport, correlates with an increased risk of developing Alzheimer's disease in individuals over 60. Mutations in presenilin genes are found in families exhibiting early-onset Alzheimer's disease, although the specific contribution of each to the Pathogenesis remains unclear. According to various reports, presenilin mutations lead to increased accumulation of Aß42. For instance, double transgenic mice obtained by crossing mice carrying the full-length human APP gene with those carrying a mutant presenilin 1 gene exhibited Aß42 overproduction. Although the pathogenesis of Alzheimer's disease is still poorly understood, it is hoped that animal models will help answer several critical questions regarding its molecular foundations. In the United States, this disease affects approximately 4 million people annually, with associated economic costs estimated at around 100 billion dollars.

Fig. 19.11. The genetic construct known as the PDAPP minigene, used to model the development of Alzheimer's disease in transgenic mice. 1–10 represent the exons of the amyloid precursor protein cDNA, and A–C denote the inserted introns. The regulatory elements include the platelet-derived growth factor ß-chain gene promoter and the SV40 virus polyadenylation signal.

Transgenic mice have also been utilized as model systems to study the expression of genes encoding transgenic products secreted into milk. For example, investigating the Functions of the protein whose defect causes cystic fibrosis (CFTR) and developing therapeutic approaches for cystic fibrosis (CF) require large quantities of authentic CFTR protein.

Cystic fibrosis is a common genetic disorder affecting approximately 1 in 2,500 newborns in European countries. The primary effect of the defective CF gene is an alteration in the function of CFTR, which normally acts as a chloride ion channel. The resulting blockage of ion flux into and out of cells causes mucus to accumulate in the ducts of several Organs, particularly the Lungs and Pancreas. This mucus becomes a breeding ground for bacterial infections that respond poorly to antibiotic Treatment. DNA released from lysed Bacteria renders the mucus extremely viscous. The thickened mucus clogs the ducts, disrupting normal organ function and further exacerbating the symptoms of cystic fibrosis. The current life expectancy for cystic fibrosis patients is 25–30 years.

To better understand the MECHANISM OF ACTION of CFTR, sufficient quantities of this protein must be available. All known in vitro cellular expression systems have failed to provide efficient synthesis, likely due to the accumulation of CFTR within the membranes of transfected cells. This problem could potentially be resolved by continuously shedding Plasma Membranes from the host cells. In such a system, the heterologous transmembrane protein would associate with detached Plasma Membrane vesicles, significantly facilitating its concentration and purification. An analogous mechanism is utilized by mammary gland cells during Lactation to produce fat globules. These lipid droplets become encapsulated in The Plasma Membrane and are secreted into milk in this form.

To test the efficacy of this system, the full-length CFTR cDNA was inserted into the middle of a defective goat ß-casein gene, from which the region spanning the end of exon 2 to the beginning of exon 7 had been deleted (Fig. 19.12). The resulting construct contained the promoter and METABOLISM/31.html">Transcription termination signals of the goat ß-casein gene. The CFTR cDNA was integrated into the structural gene along with introns, which served to enhance transgene transcription efficiency. The ß-casein gene is actively expressed in mammary gland cells during lactation, and its product constitutes the major milk protein.

Fig. 19.12. The CFTR cDNA–goat ß-casein gene construct. The full-length CFTR cDNA is inserted between exon 2 (EX2) and exon 7 (EX7) of the goat ß-casein gene. The promoter, terminator, and exons 1, 8, and 9 (EX1, EX8, and EX9) of the casein gene are preserved.

Lines of transgenic mice carrying the CFTR cDNA under the control of ß-casein regulatory sequences have been successfully established. As expected, the milk of transgenic females contained the CFTR protein associated with fat globule membranes. No adverse side effects were observed in lactating CFTR-transgenic females or in pups nursed on their milk. The CFTR protein was glycosylated and readily extracted from the milk fat fraction. It remains to be determined whether this protein is structurally and functionally authentic. The feasibility of producing other membrane-bound proteins in milk has also been investigated. Mammary gland cells of transgenic mice synthesize numerous medically relevant proteins during lactation. However, to produce CFTR, other transmembrane proteins, and various human proteins in large quantities, the corresponding transgenic constructs must be integrated into The Genome of larger mammals, such as cows, sheep, or goats.