Question

Before proceeding, you wish to know whether this gene is actually expressed in humans (your target market for therapeutic development). ?

Describe, in detail, 2 efficient experimental approaches to answer this question.

Discuss whether your proposed methods measure synthesis or accumulation of the product.

Answer 1

A number of methods are available to study the expression of genes in living animals after it has been transferred. However, of the available options, imaging techniques seems to be the best fit. However, imaging technique is also a method that uses various different elements.

Imaging techniques:

We are quite exposed to studies with reporter gene studies involving green fluorescent protein and luciferase etc. The problem with such techniques is that, they are reliable only in case of microbial studies or for small animals. In case of humans or any higher animals, what is better suited is magnetic resonance imaging (MRI) or positron emission tomography (PET). While MRI is good for high resolution tomographic images, it lacks the high-sensitivity that PET offers. After a successful vector transfer, PET is the best to monitor gene expression, even at the very lowest level and it also shows the distribution across regions of expressed genes. The idea is not to study the synthesis of gene product, but to quantitate non-invasively the amount of product formed in the body after transgenesis. PET based receptor-ligand system has been shown to work well in association with adeno-associated virus mediated gene expression for understanding/monitor the later distribution, magnitude and duration.

DNA-Liposome based gene transfer and analysis:

DNA-Liposome complexes are mixed with a solution that will aid the transfer along like lactated Ringer’s solution, in a plasmid DNA vial. The anaesthetised patient in injected with DNA-liposome complex using a 22-gauge needle (melanoma nodule, when in case of cancer studies, or pulmonary artery, or right femoral vein by right heart catheterization). Further to this, gene expression can be analysed by various methods including:

1. Biochemical and hemodynamic monitoring: basic analysis of vital signs and cardiac rhythm are involved in this, along with biochemical, hemodynamic and haematological factors

2. Analysis of the cancer gene (recombinant) expression: to confirm recombinant gene expression, genomic DNA can be recovered from biopsy. Primers are generated, differently for RNA analysis and plasmid in blood test. PCR and southern blotting is done to confirm the gene presence.

3. Immunologic analysis: CTL functions could be assayed against melanoma cells; even compared to Epstein-Barr virus transformed- B cells

In methods like these, one can understand if the gene has been successfully inserted and also have a clear monitoring of the production/synthesis of the desired product, rather than just an idea of how much has been accumulated over time.