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Breeding Scheme and Selection of Animals for DiaComp Experiments

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It is extremely important to measure quantitative parameters (e.g. plasma glucose, urinary albumin, etc.) in a way that minimizes the effects of any adventitious variables, such as seasonal changes, dietary differences, any differences in the degrees of backcrossing, or unplanned differences in genotypes. This means that, whenever possible, mice of the critical genotypes should be obtained from genotypically identical matings carried out at or close to the same time. Archival data are not appropriate.

The breeding scheme to generate the experimental animals can vary in complexity, depending on the fertility of the two genders, whether the Testgene is to be studied in homozygotes or in heterozygotes, and whether the diabetes is induced genetically or by STZ. However, it is important to remember that, when looking for the effects of the Testgene genotype on diabetic complications, the aim is to compare the Testgenemutant/mutant (or Testgenemutant/wt) diabetic animals with their diabetic litter mates that are wildtype at the Testgene locus (Testgene wt/wt).

When testing the effects of homozygosity for a mutant (such as a knockout) Testgene on diabetes induced by the dominant Ins2 Akita mutation, a conservative and usually trouble-free breeding scheme is:

Testgene mutant/wt & Ins2 wt/wt female (Inbred1) x Testgene mutant/wt & Ins2 Akita/wtmale (Inbred2)

Males and females of six genotypes result -- the three Testgene genotypes, each with or without diabetes:

  1. Testgene mutant/mutant : Ins2 Akita/wt (Inbred or F1)
  2. Testgene mutant/wt : Ins2 Akita/wt (Inbred or F1)
  3. Testgene wt/wt : Ins2 Akita/wt (Inbred or F1)
  4. Testgene mutant/mutant : Ins2 wt/wt (Inbred or F1)
  5. Testgene mutant/wt : Ins2 wt/wt (Inbred or F1)
  6. Testgene wt/wt : Ins2 wt/wt (Inbred or F1)

Note that both parents, although inbred, are heterozygotes for the mutant form of the Testgene, which are almost always healthier and have more offspring than homozygous mutants. The offspring can be F1 hybrids (genetically as uniform as inbreds but hardier), or can be kept inbred if this is preferred. Note also that this mating produces the essential genotypes (usually 1 & 3) as littermates. Studying all six genotypes is very informative, but not essential. However, if Testgene heterozygotes are included (1,2, & 3), the functional consequences of different levels of expression of the Testgene on diabetic complications can be determined. If wildtype and mutant Testgene mice are studied on both diabetic and non-diabetic backgrounds (1,3,4 & 6), additive, super-additive or sub-additive interactions between the Testgene and diabetes can be detected.

In the case of diabetes induced by the Akita mutation in heterozygous state, it has been the DiaComp experience (1) that a higher level of chronic hyperglycemia is seen in males, and (2) that the development of diabetic complications is age-dependent and largely male-limited. Thus, the investigator could cull females as soon as sex can be determined and wean only males, thereby reducing costs and saving colony space.

When testing the effects of transgenes, it is generally advisable to avoid homozygosity since undesirable artifacts (insertional mutagenesis leading to homozygous lethality or other major physiologic/metabolic anomalies) can result independent of the presence or absence of diabetes.

When homozygous transgenes are not needed, or when the homozygous knockout of a Testgene is lethal, a simpler breeding scheme can be used:

Testgene mutant/wt & Ins2 wt/wt female (Inbred1) x Testgene wt/wt & Ins2 Akita/wt male (Inbred2)

Males and females of four genotypes result:

  1. Testgene mutant/wt : Ins2 Akita/wt (Inbred or F1)
  2. Testgene wt/wt : Ins2 Akita/wt (Inbred or F1)
  3. Testgene mutant/wt : Ins2 wt/wt (Inbred or F1)
  4. Testgene wt/wt : Ins2 wt/wt (Inbred or F1)

We append recent results that Dr. Nobuyuki Takahashi has obtained in an experiment to determine the effects of Nos3 genotype on diabetic nephropathy. He studied all six male genotypes resulting from Nos3 +/-Ins2 wt/wt female (129SvEv) x Nos3 +/- Ins2 Akita/wt male (C57BL/6) matings. The results with the resulting F1 mice are internally very consistent, and the error bars are relatively small. Interestingly the phenotypes of all six genotypes are distinguishable and progressively more severe in going from 6 to 1.


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