On How to Build a "Knockout" Mouse - (Feb/25/2015 )

If you want to study how a particular gene, named XY, might play a role under certain condition. You may study mice that are missing this gene. To "knockout" the XY gene, you will replace it with a mutated copy that doesn't work.

Here is the procedure:

1. Isolate Stem Cells

Isolate embryonic stem cells that originated from male brown mice with a normal XY gene.


2. Add Inactive Gene With Marker

To these cells, add a copy containing a mutated, inactive OhNo gene, and a drug resistance marker gene.


3. Similar Genes Naturally Swap

By mechanisms that are not completely understood yet, similar genes will swap places. The XY gene plus drug resistance marker gene is incorporated into the genome, and the normal version is kicked out. This process is called homologous recombination.


4. Add Drug

Cells that haven't incorporated the inactive XY gene don't have the drug resistance marker gene.

Adding the drug kills cells without the marker, leaving you with only cells that have an inactive version of the XY gene.


5. Grow Chimeric Mice

By transplanting stem cells that carry the inactive XY gene into a white mouse embyro, you'll create what is called a chimera. Chimeras have patches of cells throughout their bodies that grew from white mouse cells and patches that grew from brown stem cells. Some of the cells that have the inactive XY gene may develop into reproductive cells.


6. Mate Male Chimera

If a male chimera has some reproductive cells (sperm) that originated from the brown stem cells, he will produce some brown offspring when mated with a white female.


7. Test and Breed Brown Offspring

Half of the brown offspring will have a copy of the inactive XY gene in all of their cellsincluding their reproductive cells. These mice have one normal copy of the XY gene from their mother and one inactive copy from their father. So half of their reproductive cells will contain a normal copy, and half will contain an inactive copy.

These mice can be identified by performing DNA sequencing in their XY genes and then bred with each other.


8. You've Made a Knockout Mouse

One fourth of your resulting offsping will have two copies of the "knocked-out" or inactive XY gene. You can now study these mice to determine how lacking the XY gene may affect their behavior.

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