JAX Notes June 01, 2006

The importance of genetic background in mouse-based biomedical research

You may occasionally see the following cautionary note on strain data sheets of some of our JAX® Mice models:

This strain is on a genetic background different from that on which the allele was first characterized. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

We include this note because the variety of genetic backgrounds and the mutations characterized and published on them are continually increasing. As a result, researchers must be more mindful than ever of the genetic backgrounds of the mouse models they use. This article defines genetic background, explains why its influence in mouse-based biomedical research must be seriously considered, and prescribes practices for minimizing its potentially confounding effects.

Genetic background: definition and examples

As applied to a mutant mouse strain, genetic background refers to its genetic make-up (all its alleles at all loci) except the mutated gene of interest and a very small amount of other genetic material, generally from one or two other strains. As we shall see, that "other" genetic material can significantly influence a mutant strain's phenotype. Correct strain nomenclature indicates what a mutant strain's background is.

For example, the genetic background of the targeted mutant strains NOD.129S7(B6)-Rag1tm1Mom/J (003729) and NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/SzJ (004848) is primarily NOD. However, the first strain carries a targeted mutation of the Rag1 gene, likely a few Rag1-linked alleles from 129S7-derived ES cells, and possibly some B6 alleles from crosses in its breeding history. In contrast, the second strain is a congenic (Cg) with more than one donor strain in its breeding history. It carries targeted mutations of the Rag1 and Prf1 genes and possibly some background alleles from those other strains.

Similarly, the genetic background of transgenic strains FVB/N-Tg(MMTVneu)202Mul/J (002376) and FVB/N-Tg(MMTV-PyVT)634Mul/J (002374) is FVB/N. However, whereas the first strain carries an MMTVneu transgene, the second carries an MMTV-PyVT transgene. On the other hand, strains B6.129S7-Rag1tm1Mom/J (002216) and NOD.129S7(B6)-Rag1tm1Mom/J (003729) each have the same targeted mutation of the Rag1 gene, but whereas the first is on the C57BL/6J (B6) background, the second is on the NOD background.

Genetic background does make a difference

The technology for producing genetically engineered mice has been substantially refined, resulting in an ever-increasing number, variety and availability of mutant mouse models. Generally, alleles of interest (such as spontaneous mutations, targeted mutations, transgenes and congenic regions) may be maintained on one to several backgrounds that are more vigorous, better characterized, more amenable to scientific experiments, reproduce better, display a phenotype better than, or have some other advantages over other backgrounds.

However, these alleles are sometimes transferred to backgrounds that are not well characterized. In any case, inattention to a mutant's genetic background can seriously confound research results. Each strain has unique background alleles that may interact with and modify the expression of a mutation, transgene or other genetic insert. The likelihood of such modifier genes having a confounding effect is especially high in an uncharacterized background or in a segregating or mixed background of unspecified origin. Even in a well-characterized strain, undiscovered modifier genes may confound results, sometimes making them unexplainable. Such modifier genes are the reason why normal development and physiology often vary significantly among inbred strains.

One of the first documented instances of the influence of genetic background on gene expression was the discovery that, on a B6 background, the diabetes (db) and obese (ob) mutations causes obesity and transient diabetes. But on a C57BLKS/J (BKS) background, they cause obesity and overt diabetes (Coleman and Hummel 1973; Coleman 1978). Those results indicated that background-unique modifier genes were influencing the expression of the ob and db mutations.

Since then, many other examples of background-unique modifier genes have been shown. They may influence gene expression by suppressing or enhancing it, altering DNA transcription rates or mRNA stability, and inducing epigenetic effects, such as DNA methylation. Their effects have been most well documented in mice that harbor targeted mutations (knockouts), congenic regions and transgenes.

Modifier genes may be unique in the background strains that harbor these genetic alterations, or they may be inadvertently introgressed into the background strains in the process of constructing a congenic (in the latter case, they may either be linked to or independent of the introgressed locus). They may also arise spontaneously, as the modifier of Min 2 (Mom2) mutation reportedly did (Silverman et al. 2002). Following are a few of the many published examples of the effects of genetic background on gene expression:

The multiple intestinal neoplasia mutation (Min) of the adenomatous polyposis coli (Apc) gene (ApcMin). B6 mice heterozygous for the ApcMin mutation are very susceptible to developing intestinal polyps. Offspring of these mice mated with AKR/J, MA/MyJ, or CAST mice are significantly less susceptible, indicating that the latter three strains harbor strain-unique ApcMin modifier loci, named modifier of Min 1 (Mom1) (Moser et al. 1992; Dietrich et al. 1993). The AKR allele of Mom1 has been shown to actually contain two genes (MacPhee et al. 1995; Cormier et al. 1997, 2000).

Here are other examples:

  • The Alzheimer's amyloid precursor protein transgene, Tg(APP695). In outbred mice, this transgene merely induces the formation of amyloid plaques in the brain; in inbred FVB/N and B6 mice, it is lethal (Carlson et al. 1997).
  • The Prkdcscid mutation. In the NOD background, the Prkdcscid mutation is associated with low natural killer (NK) cell activity, no complement activity, impaired macrophage development and impaired antigen presenting cell functions; in the BALB/c background, it is associated with high NK cell activity, high complement activity, normal macrophage development and normal antigen-presenting cell functions (Custer et al. 1985; Shultz et al. 1995).
  • Prkdcscid-associated leakiness (tendency to produce some functional B and T cells with age). Leakiness in Prkdcscid mutants is generally high on the B6 and BALB/c backgrounds, low on the C3H background and very low on the NOD background (Shultz et al. 1995).
  • IL10-deficiency. In the B6 background, IL10-deficiency only slightly increases susceptibility to inflammatory bowel disease (IBD); in the 129/SvEv, BALB/c, and C3H/HeJBir backgrounds, it greatly increases IBD susceptibility (Beckwith et al. 2005).

Many phenotypic differences among substrains can only be explained by as yet undiscovered background-unique modifiers, most likely spontaneous mutations. Following are several examples:

  • Differences in tissue rejection among related 129 strains (Simpson et al. 1997).
  • Differences in susceptibility to proteoglycan-induced arthritis and spondylitis among C3H substrains (Glant et al, 2001).
  • Differences in fear responses between C57BL/6J and C57BL/6N substrains (Radulovic et al. 1998; Stiedl et al. 1999).
  • Differences in the effects of anesthetics on cardiac function between C57BL/6J and C57BL/6N substrains (Roth et al. 2002).

The ability of background-unique modifiers to confound research results is not merely a theoretical possibility. Published reports indicate that it has already happened. These reports include wasted efforts due to a mix-up in AL/N substrains (Bailey 1982), confounded results due to effects of genetic background differences among 129 substrains (Threadgill et al. 1997), and dubious results because of different genetic background effects among C57BL substrains (Specht et al. 2001; Wotjak 2003). Many such effects have likely not been reported or discovered.

Tips to minimize the confounding effects of genetic background

Although you may not be able to eliminate the confounding effects of genetic background in your research, you can minimize them considerably by observing the following practices:

  • Use mutants with genetically well-defined backgrounds (Silver 1995; Linder 2006, 2001; Yoshiki and Moriwaki 2006).
  • Use appropriate controls. If a mutation arose spontaneously or was induced on a well-characterized inbred strain, the inbred strain is likely coisogenic with and therefore the best control for the mutant harboring that mutation (Silver 1995; Linder 2001, 2006).
  • When possible, construct congenic, targeted mutation, transgenic and other genetically altered strains that are coisogenic to controls (Silver 1995; Linder 2006).
  • Construct congenics and transgenics on well-defined backgrounds, such as B6 and FVB (Silver 1995; Linder 2006).
  • Construct targeted mutation strains on well-defined ES cell lines, derived either from B6 or well-defined 129 strains (Simpson et al. 1997; Threadgill et al. 1997; Linder 2006).
  • If possible, analyze mutations on several backgrounds. If the mutation is maintained on a mixed genetic background, analyze it in hybrids of the two progenitor strains (Silver 1995; Linder 2001, 2006).
  • Consider the effects of environmental factors such as noise, light, home cage environment, handling and diet on gene expression and behavior (Crawley et al. 1997; Bailey et al. 2006).
  • In all your research communications, describe your mouse models with correct nomenclature (Linder 2006).

In summary, the importance of considering the genetic backgrounds of mouse models used in biomedical research cannot be over-emphasized. If that research is to be reliable and reproducible over time and place, and, more importantly, if it is to have the most potential for improving human health, it must be conducted with models whose genetic backgrounds are well-defined, stable and clearly communicated.

References

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Bailey KR, Rustay NR, Crawley JN. 2006. Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls. ILAR J 47:124-31.

Beckwith J, Cong Y, Sundberg JP, Elson CO, Leiter EH. 2005. Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens. Gastroenterology 129:1473?84.

Carlson GA, Borchelt DR, Dake A, Turner S, Danielson V, Coffin JD, Eckman C, Meiners J, Nilsen SP, Younkin SG, Hsiao KK. 1997. Genetic modification of the phenotypes produced by amyloid precursor protein overexpression in transgenic mice. Hum Mol Genet 6:1951-9.

Coleman DL. 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14:141-8.

Coleman DL, Hummel KP. 1973. The influence of genetic background on the expression of the obese (Ob) gene in the mouse. Diabetologia 9:287-93.

Cormier RT, Bilger A, Lillich AJ, Halberg RB, Hong KH, Gould KA, Borenstein N, Lander ES, Dove WF. 2000. The Mom1 AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64. Oncogene 19:3182?92.

Cormier RT, Hong KH, Halberg RB, Hawkins TL, Richardson P, Mulherkar R, Dove WF, Lander ES. 1997. Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis. Nat Genet 17:88?91.

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Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo C, Borenstein N, Dove W. 1993. Genetic identification of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell 75:631?9.

Glant TT, Bardos T, Vermes C, Chandrasekaran R, Valdez JC, Otto JM, Gerard D, Velins S, Lovasz G, Zhang J, Mikecz K, Finnegan A. 2001. Variations in susceptibility to proteoglycan-induced arthritis and spondylitis among C3H substrains of mice: evidence of genetically acquired resistance to autoimmune disease. Arthritis Rheum 44:682-92.

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Linder CC. 2001. The influence of genetic background on spontaneous and genetically engineered mouse models of complex diseases. Lab Anim 30:34-9.

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Silverman KA, Koratkar R, Siracusa LD, Buchberg AM. 2002. Identification of the modifier of min 2 (Mom2) locus, a new mutation that influences Apc-induced intestinal neoplasia. Genome Res 12:88-97.

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