Gene Knock-out

Timeline:
Gene Knock-out was first discovered in 1987 by Dr Mario Capecchi of University of Utah
alongside his colleagues Oliver Smithies of University of North Carolina, Chapel Hill, and
Sir Martin Evans of Cardiff University, UK where they received in 2007 the Nobel Prize in
Physiology or Medicine for developing the technique.
It all started when the embryonic stem cell of the mouse was identified by Martin Evans and
was isolated from the early stages of the embryo. Established in cell culture, it was then
genetically modified and reintroduced into foster others for the generating the genetically
modified offspring. Mario Capecchi and Oliver Smithies, then discovered how homologour
recombination between segments of DNA molecules used to target genes in the mammalian
genome and developed methods to produce genetically modified mice. (Rajashekar et. al,
2013)
What is the purpose of the technique?
The main objective of creating a knock-out is to eliminate or cause a disturbance of a specific
gene or genes of interest and finding out the effects of it towards the organism. Thus, causing
physical, behavioural and biochemical changes if there is any or any other metabolic
phenotypes or functions observed in the knock-out (KO) animal. From there, then one is able
to study the gene of interest which has some functional role or roles through phenotype
observations. This technology has been experimented on mice which led to similar
technology on zebrafish, rats, pigs, fruit flies and etc. KO plants are also active in this area of
research.

Procedure:
LAB PROCEDURE DONE BY THE RESEARCHER
A. Making the DNA construct [in a span of 6 months to complete]
1. Acquire the genomic DNA for your gene or locus of interest. You can either screen a
129/Sv genomic library or use genomic databases, public bacterial artificial
chromosomes (BACs) and PCR for a C57BL/6 background.

This process takes about 1-3 months

2. Make a DNA construct.
DNA from the genomic locus (orange) flanks the DNA to be inserted (blue) and it
is placed in a bacterial plasmid. This construct is designed to add new pieces of
DNA into the mouse genome at a desired locus. In a knockout experiment, the
new DNA will replace the normal gene. Linearize the construct and send to the
manufacturer to be cut.

This process takes about 3-6 months.

DONE BY THE MANUFACTURER
B. Generating Targeted ES cells [in a span of 3 weeks]
1. Insert the DNA into embryonic stem cells (ES cells) via electroporation.

2. In the cells, the homologous pieces of DNA recombine. This inserts new DNA from
the construct into the desired locus, usually replacing a portion of the original
genome.

3. Place the ES cells in selective media, allowing for the growth of cells containing the
DNA construct.
- The DNA construct has a drug-resistance marker
- Very few of the cells take up the construct cells that do not will die because
they are not resistant to the drug added to the media.

4. Up to 500 isolated colonies are isolated and expanded in plates.

5. Isolated DNA from individual colonies is shipped back to the researcher and the ES
cell stocks are frozen.
IN THE RESEARCHERS LAB
6. Southern blotting or PCR is used to determine which colonies have integrated the
DNA at the correct locus. Targeted recombination is generally rare, so only a few
clones will usually be correct

The manufacturer will send an email informing the researcher which colonies should
be used to make mice.

C. Making the Mice

1. Culture ES cells containing the correct genetic modification.

2. Brown ES cells are then injected into black mouse blastocysts (3.5 day old embryos).
The ES cells with the modification will contribute to a percentage of the embryos
tissues.
3. Transfer injected blastocysts to the uterus of a recipient of a mouse which will act as a
surrogate mother.

4. Pups containing tissues from the modified ES cells are called chimeras The donor
embryo and the ES cells are from different strains of mice with different coat colours
(black and brown). This allows you to visually select chimeras they look like striped
mice.

5. Breed 8 week old chimeras to females with black coats.

6. If any brown offspring are born, the researcher knows the modified ES cells have
contributed to the germline (sperm) of the chimera.

7. Genetic testing is performed on the offspring to determine which mice carry the
genetic modification.

Source: MIT Aurora Burds Connor February 2007 - Schematic and Time Line for the
Generation of Knockout Mice

Reference
Schematic and Time Line for the Generation of Knockout Mice. (February 2007). Retrieved
from: https://ki.mit.edu/files/ki/cfile/sbc/escell/timeline-complete.pdf

Rajashekar, B. et al (2013). Gene Kicked Mouse: Knock Out Mouse and Its Application,
International Research Journal of Pharmacy. 4(7).
Klug, W. S. et. al (2016). Concepts of Genetics: Eleventh Edition. London: Pearson
Education.