Question

Design an experiment to knock out a certain gene of interest in the human genome. Outline the experiment, explaining the methods you would use, and also indicate how you would verify your experiment was successful at each step.

Answer 1

Gene knockout method or gene targeting method is used to study the function of a gene. It is obtained either by removing exons, deleting a gene and introducing point mutation. Gene targeting uses homologous recombination strategy to change an endogenous gene.

The gene from which a DNA sequence has been removed or altered is inserted into the germline cells or stem cells of the organism and allowed to grow. Then this can be inserted into the growing embryo using artificial vectors.

The result of gene knockout experiment will be visible as a phenotypic variation or as an alteration on biochemical expression.

Methods in process of gene knockout:

1. Selection of gene for knockout study:

In case of human gene knockout suppose a gene HEC that result in eye color of human. To study its activity it has to be suppressed or inactivated. Besides the structure of gene its total length and other information needs to be obtained.

This can be performed by introducing a mutation in the DNA sequence of the gene or inactivate the promoter sequence of the gene that regulate its expression or can remove the entire gene from the DNA sequence.

2. Selection of vector:

Vector is used to transfer gene of interest to target cells. A plasmid is generally used as a vector for gene transfer. Most commonly used vector is Bacterial Artificial Chromosome (BAC) for gene knockout experiments.

If we have removed the gene sequence for studying its function or introduced a mutation into the DNA sequence and inserted this altered gene into the vector. Along with this a marker gene is also introduced into the vector for confirming our insert into the vector.

3. Plasmid insertion into the embryonic stem cells:

Once the vector is ready with the gene of interest, it is then inserted into the embryonic stem cells through methods as electroporation, sonication or microinjection.

The plasmid when inside stem cells and find its target gene, process of recombination occurs and the mutation is placed into the target gene.

The transformed cells are then grown in the selective media that allows growing of cells containing the marker gene. This helps in differentiating the cells which contains the gene of interest from the cells which have normal cells.

4. Confirmation of the gene insert using PCR:

To confirm that cells have inserted gene sequence polymerase chain reaction is performed. DNA is extracted from the cultured cells and with the primers that are specific to DNA sequence are used for PCR amplification.

If there are amplicons it confirms the transformation of cells.

In this manner embryonic stem cells are transformed and these are now termed as genetically modified cells.

5. Injecting into the embryo and breeding:

To understand the gene knockout transformed cells are inserted into the model organism embryo. The embryo cell has two populations normal and transformed. This is termed chimeric organism. This is then bred with normal organism where it produces offspring with two different types of genotypes: normal and altered.

6. Gene knockout examination:

Once gene knockout organism is ready it is examined for gene parameter through PCR amplification method and experimental results can be validated.

In the PCR reaction if the target gene recombined with the antibiotic marker gene and a DNA band is obtained with the primer sequence that means the transformation was successful. In case of non-transformed cells with primers do not result in DNA band. A control with wild type DNA is used for comparison of the results.

This is further confirmed by running PCR samples on 2 % agarose gel electrophoresis.