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Molecular Genetics of Human Diseases
Essay plan
Describe chemical/physical and biological methods for gene transfer into
mammalian cells, including those used for gene therapy
Introduction
Gene transfer: the insertion of unrelated genetic information in the form of DNA into cells
(Roberts, 2012)
Why gene transfer:
- treatment of diseases using gene transfer to supply patients with therapeutic genes
- different methods: chemical and physical; biological – viral and non-viral
- viral vectors: viruses that are genetically altered to still enter cells or express genes
without replicate and become infectious; commonly used in gene therapy
Gene therapy: deliberate transfer of DNA for therapeutic purposes
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Inherited disorders in which a single
gene is missing or abnormal
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The gene must be properly
expressed in sufficient quantities
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Add medium and apply to cells
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ADA)
- Gene-corrected cells then injected back to patient
Advantages & disadvantages:
- single copy integration of DNA
- broad host range: numerous clinical trials are using retroviral vectors
- have the ability to transfer DNA to a high fraction of cells, but using virus as the
vector will potentially arouse cancer leukemia (Cavazzana-Calvo, 2004)
- carry only 8-9kb of DNA

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infect only growing cells
retroviral DNA random integrates

Gene therapy for cystic fibrosis
- CF is an autosomal recessive disorder
- 1/2500 in white male population
- gene coding for CFTR (cystic fibrosis transmembrane conductance regulator) was
transferred into epithelial cells of the lungs of CF patients
- used adenovirus-derived vector
- problem: severe immunoresponse
- adenovirus vectors: DNA containing viruses, infect many types of growing and nongrowing cells
- for gene therapy: two early genes E1 and E3 are deleted and replaced with gene of
interest
- gene injected into viral vector, to be introduced into a human cell
- Adenovirus vectors:
- Advantages: broad host range (both dividing and non-dividing cells)
- Disadvantages: DNA does not integrate into the host genome, remains episomal
- Patients develop neutralizing antibodies against viral proteins

Conclusion
Despite the advancement of technology nowadays, gene therapy is not yet effective
for treatment in genetic disorders
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Besides that, there have been
problems with viral vectors in a way there is toxicity, immune and inflammatory responses,
and gene control and targeting issues
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In the future, if researchers put together more effort in trial-and-error, there is a
possibility for better developments in gene therapy
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40,000 cells placed per well of a 24-well plate in 0
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Cells incubated at 37°C in a CO3 incubator until the cells are 50-80% confluent
(about 20 hours; depending on cells)
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Dilute 3µl Lipofectamine reagent into 25µl medium without serum for each well and
mix
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In this step the DNA liposome complexes are formed
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2ml transfection medium without serum
7
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15ml medium without serum to the tube containing the complexes for each
well
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The incubating time will be flexible by the cell type
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4ml growth medium containing double the 2x normal concentration of the
serum without removing the transfection mixture
10
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Assay cell extracts for transient gene expression at 24-72 hours after transfection,
depending on cell type and promoter activity
12
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Trying to memorise



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