Animal Model Selection
Selecting your Model
While every application of chimeric animals is unique, we would like to offer some general guidelines for model selection based on our internal experience. This is not a guarantee of performance for your particular application.
Human Repopulated Animals
Which Strain Should I Use?
In our hands, the C57Bl/6 and NOD background perform similarly for all applications. More data and validation have been completed on the C57Bl/6 strain, so this is a consideration. However, the NOD strain is slightly larger which provides an advantage in applications where multiple blood samples are required from a single animal. If you envision that your application will change over time and evolve to include an immune system element, we would recommend that you start with the NOD background to establish your baseline data with the same strain.
What Level of Repopulation is Right for Me?
There are a multitude of factors that impact this decision including application, compound selection, and target specificity. In general, we would say that the higher the level of human repopulation, the better the result is likely to be. However, in many instances we have found that the application is sufficiently addressed at a lower level of repopulation, which can save wait time and money. In any case, proper controls are essential to being able to interpret your data and we recommend that your consult the literature and our staff to aid you in your final selection.
Humanization of 70%+
We’ve had tremendous success using the FRG™ KO for a variety of human infectious diseases including hepatitis B & C and malaria. Depending on the pathogen and the titer, the total percentage of cells infected is highly variable, which means that the infection becomes an exercise in probability. For hepatitis C and malaria, which have lower rates of infection, we generally recommend using animals that are greater than 70% repopulated. For hepatitis B, animals that are humanized to 40-70% work very well.
While this area is undoubtedly the most published use for chimeric systems, it is also the most problematic in terms of application and interpretation. Before proceeding in this direction, please consider the following questions:
- Is the compound primarily metabolized by hepatocytes?
- Is the contribution from intestine and kidney understood?
- Have differences in clearance been observed between human and mouse?
- Is a difference in metabolism observed according to route of administration – intravenous as compared to oral?
If the compound is metabolized primarily by the hepatocytes and is cleared similarly between mouse and human cells, it is likely that you will have reasonable results with chimeric systems. If your compound is primarily metabolized by the kidney and is preferentially metabolized by the mouse, it is not likely that human results will be recapitulated.
At this point in the field, it is too early to say if these tools can be used early screening of drugs, but rather can be a potent tool in an instance where a considerable amount of information is already known and the compound requires further understanding or validation. We have observed that many model compounds have been selected based on their ability to provide species-specific metabolites, only to see the compounds fail in vivo due to rapid clearance rates in rodent.
If you believe that these models are well suited to your application, we strongly recommend that you allow us to work closely with your group to design the study and controls. Our staff is highly experienced in reviewing and administering these studies, and getting their perspective can save time, money, and aggravation when the time comes to analyze data.
Like metabolism, toxicology is highly dependent on the target of interest and your compound. If your application is looking specifically at human toxicity in hepatocytes, then a model that is highly repopulated makes sense. Frequently, we utilize models to examine species-specific toxicity and desire approximately equal populations of human and murine cells, in which case we recommend animals that are in the 40-70% range.
Humanization of 40-70%
Highly promiscuous pathogens are well suited to animals in this range. We have had excellent results with hepatitis B and are able to maintain persistent infection and infectivity for periods of more than six months.
Unless your compound is specifically metabolized by human hepatocytes, we do not recommend using animals in this range of repopulation for your studies. While we could imagine scenarios where human metabolic recapitulation would be possible, we have not yet encountered one.
Animals that are roughly 50% humanized have been used in species-specific toxicology probing with good results. For compounds that have demonstrated rodent toxicity, the system can be utilized to demonstrate that the toxicity is rodent-specific. For compounds that have demonstrated human toxicity in the clinic, the model is used to explore toxicity that was missed in rodent testing.
Humanization Below 40%
For those that are looking to species-specific specific targeting of gene therapy vectors, these animals are well suited to the application.
Mouse Repopulated Animals
What Do I Need Controls For?
Controls are an essential component of your study and are frequently overlooked in early study design. By utilizing appropriate controls, we can distinguish effects that are generated in the murine background and validate that a system perturbation is due to the human cell population.
So Which Control is Best for Me?
Controls come in a variety of flavors and should be selected according to exactly what needs to be controlled. On occasion a wild-type Rag/Gamma is a simple and efficient control for studies. However, if you are looking to control for surgery, transplantation, antibiotic treatments, and CuRx™ Nitisinone treatment, we recommend using controls that have been engrafted with mouse hepatocytes. Animals can be repopulated with the original background strain (C57Bl/6 or NOD) or with ROSA26 hepatocytes. In either instance, the animals are treated exactly the same as human repopulated animals and can be age matched. ROSA26 cells have the added functionality of being able to be stained with X-Gal to distinguish mutant Fah cells from wild-type cells.