I am trying to design a wet lab experiment with no wet lab experience to name.

Right now, in my dream land, it would be excellent if it were possible to create a Caspase-9 knockout mouse (damage to the Caspase-9 gene is associated with the development of breast cancer), and somehow be able to control the development of the breast cancer phenotype, so I can create a Caspase-9 KO control as well as a non Caspase-9 control group. In total, I would love to have the following control groups:

1. Normal mouse - normal Caspase9 gene, no breast cancer phenotype
2. Caspase9 KO mouse - no breast cancer phenotype
3. Caspase9 KO mouse - breast cancer phenotype

Can anyone help me answer whether or not group 2 above is possible? Can you direct me to any papers, any experiments, where this has been done before, or has been tried below and failed?

Here is a paper that talks about the association of Caspase-9 with breast cancer development: http://breast-cancer-research.com/content/pdf/s13058-014-0443-5.pdf. Caspase-9 is a type 2 member of the Caspase protein family. When I talk of the "Caspase-9 gene", I'm talking about the DNA which codes for these cellular apoptosis susceptibility 9 (Caspase-9) proteins. This isn't my area of expertise, but this is what I currently understand.

  • $\begingroup$ is there even a thing like Cas9 gene? As I know, cas9-CRISPR system is used to create double-stranded breaks in genome, so it can be used for targeted mutagenesis, including creating knock-outs. Please, provide reference to cas-9 gene and breast cancer association $\endgroup$ Apr 14, 2015 at 22:04
  • $\begingroup$ and there Cas9 is bacterial protein $\endgroup$ Apr 14, 2015 at 23:35
  • $\begingroup$ There seems to be a caspase 9 in mammals which is different from cas9. The question should probably be edited to make this clear. $\endgroup$
    – user137
    Apr 14, 2015 at 23:50
  • 6
    $\begingroup$ I think the paper you refer to uses cas to refer to the cas family of proteins, still not the CRISPR/Cas9. Biologists need better names. $\endgroup$
    – user137
    Apr 15, 2015 at 0:22
  • $\begingroup$ Cas9[CRISPR] totally dominates google search these days, yes. $\endgroup$ Apr 15, 2015 at 5:14

3 Answers 3


The short answer is that it is likely a bad experiment; being that it would be expensive and is not likely to provide useful new information.

The long answer (i.e. why) is identifying an interesting target gene (e.g. caspase-9 in breast cancer) is only the first step, and considerable study of what is already known is required before planning an experiment (especially an expensive one that requires numerous mice).

Caspase-9 is a critical effector of apoptosis through the mitochondrial pathway. Many different signals such as DNA damage, Endoplasmic reticulum stress, etc induce cells to die through this pathway. This is important both in normal development and in preventing cancer. Homozygous deletion of caspase-9 is lethal, the mice will not live to adulthood. (http://www.ncbi.nlm.nih.gov/books/NBK6153/) A conditional knock-out of caspase-9 in breast cancer cells would be needed, not a global deletion.

Furthermore, the given the known functions of caspase-9 this experiment is not likely to provide interesting new information. This pathway is well characterized and known to play an important role in cancer (Resisting cell death; a hallmark of cancer: http://www.cell.com/abstract/S0092-8674(11)00127-9 ) Loss of caspase-9 activity makes cells resistant to apoptotic signals through the mitochondrial pathway(they won't die when they receive these "suicide" signals). This allows mutant cells to survive when they should otherwise die. Cancer cells can also become resistant to this pathway through other means (such as the up regulation of anti-apoptotic Bcl-2 family members.

Many scientists are already studying how to restore normal function of this pathways in various cancers; what do you hope to learn by breaking it that isn't already known (I'm not saying that there couldn't be a good reason, but if you don't have one, you shouldn't do it.)

  • $\begingroup$ +1 for pointing out that the results of this proposed experiment are already known (for knockout, not knockdown) $\endgroup$
    – March Ho
    Jun 15, 2015 at 16:58

This seems to be a standard procedure knockout and complementation rescue experiment.

Breed 2 can be produced from Breed 3 (assuming the Caspase 9 gene indeed causes cancer when it is not expressed).

Firstly, knockout Caspase 9 from a wild type, creating Breed 3. Verify that the cancer phenotype is shown and is statistically significant.

Then, transform Breed 3 with an artificial copy of the Caspase 9 gene in an alternative chromosomal location. This breed (Breed 2) will act as a positive control for the gene, and should behave identically to the wild type.

Finally, you can then produce multiple breeds of mice transformed with different versions of the gene (for example serine/threonine mutagenesis to detect the kinase specificity) in order to determine mutations that cause the Caspase 9 protein to become non-functional. Alternatively, the same gene but with a stronger/weaker promoter can be used to determine the dose-dependent effects of the gene.

This paper regarding a circadian clock gene shows the general procedure required to carry out such a complementation rescue experiment.

  • $\begingroup$ If you transfom a previously knocked out gene, then it is not a knock out anymore since it has functioning copy of the gene... At least my logic dictates this. $\endgroup$ Apr 15, 2015 at 18:36

It seems you are confused with some other gene instead of caspase 9, but if KO of some gene causes development of breast cancer, and you want to compare something between none-cancerous and cancerous states, you could take several ways:

  1. See if you could have littermates carrying and not carrying cancer. You could compare them. Sometimes, phenotype do not express 100% among mice through the life spam or at younger age.

  2. Breast cancer is rare in human males, probably in murine males, too; think you could compare what you want to see between males and females.

  3. When you cross KO mice with other strains, occurrence of phenotype could vary. Then you could compare what you want to see between less and more susceptible mice strains.


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