Question

Describe knockout mouse and dont forget to mention the viral vectors and DNA markers and their use.

Answer 1

A knockout mouse is an genetically modified mouse where a gene is inactivated and then replaced by an artificial gene

Steps to produce a knockout mouse

First step to produce knockout mice, researchers use one of two methods to insert artificial DNA into the chromosomes contained in the nuclei of ES cells. Both the methods is carried out in vitro, that is in cultured cells grown in laboratory conditions.The conditions are invitro

When it comes to making knockout mice, the first and most important part is the design of a targeting vector. The first step is to identify the gene that has to be “knocked out,” then use specific markers in order to facilitate the preparation of the targeting vector. Upon identifying the gene and its flanking DNA sequences, markers such as neo and TK are used to create the targeting vector. The vector is then inserted into the ES cells through the use of a process known as electroporation

The next step in making knockout mice is an extremely important one. The cells that were modified through electroporation now have two options on how to respond. One group of cells will allow the targeting vector to recombine with the gene that was targeted, which leads to one copy of the gene being knocked out in those cells. Another group will involve the vector recombining in the wrong locus, which means these cells cannot be used. To separate the correct cells from those that have to be discarded, researchers typically use a drug-resistant marker. This marker will make successfully targeted ES cells immune to certain drugs, which can then be used to kill off the cells that don’t have the marker.

Chimeric Mouse creation

Once the right cells are prepared, it’s time to inject them into the blastocyst that will become the target chimeric mouse. The ES cells will typically be transplanted into the healthy blastocyst contained inside the uterus of a white mouse. Making knockout mice this way is convenient, since the resulting mouse will have white and dark patches, making it easily identifiable as a chimera.

Breeding Chimeric Mice

And Once the chimeric mouse is successfully generated, the final process is to test it and breed it. The chimeric mouse will normally be bred with a normal, white mouse, in order to pass down the altered genetic material to its offspring. Typically, if the experiment is successful, the resulting mice will have a healthy version of the knockout gene from their mother and a knocked out version of the gene resulting from their father. These results will become visible when performing DNA sequencing on the gene in question. As scientists become more proficient at making knockout mice on a regular basis, they’ll learn specific techniques for targeting genes more successfully and increasing the rate of success in every step.

These are the steps in making na knockout mouse it is important to note that all the procedures are vitro.