Brief Reviews |
From the Departments of Internal Medicine and Physiology & Biophysics, The University of Iowa College of Medicine, Iowa City.
Correspondence to Curt D. Sigmund, PhD, Chair, Molecular Biology Interdisciplinary Program, Director, Transgenic and Gene Targeting Facility, Department of Internal Medicine and Physiology & Biophysics, 2191 Medical Laboratory, The University of Iowa College of Medicine, Iowa City, IA 52242. E-mail curt-sigmund{at}uiowa.edu
| Abstract |
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Key Words: transgenic mice knockout mice genetics epigenetic
| Introduction |
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Studies performed over the past few years have clearly illustrated that
phenotypes caused by specific genetic modifications are
strongly influenced by genes unlinked to the target locus. For example,
whereas deletion of the p53 tumor suppressor gene causes a dramatic
increase in the frequency of tumor formation in those mice compared
with wild-type mice, the types of tumors formed, their numbers per
animal, and age of tumor onset vary in different genetic
backgrounds.1 2 3 Other phenotypes observed in
transgenic and gene-targeted animals influenced by genetic background
include ethanol tolerance, sepsis, immunity, locomotor activity,
behavior, organ structure, development, and
cardiovascular physiology
(Table
). As examples of the
latter, the incidence of stroke in mice deficient in tissue
plasminogen activator and susceptibility to
atherosclerosis in apoE-deficient mice differ when the
knockout loci are present on C57BL/6, 129/Sv, or FVB/N
backgrounds.8 13
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On a positive note, phenotypic differences caused by allelic variation outside the target locus can provide a molecular genetic tool to identify and clone "modifier genes," which influence a phenotype.17 However, as stated above, these differences can cause significant problems when interpreting and comparing the results of transgenic and knockout studies between laboratories.
| The Problem |
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A second issue specifically related to transgenic mice (but not gene-targeted mice) is the position effect. Because of the random nature of the transgene insertion event after pronuclear injection, each resultant founder contains the transgene at a different site in the genome. These position effects can profoundly influence transgene expression and, therefore, the observed phenotype.20 21 This occurs because transcriptional regulatory elements present at or near the site of insertion (controlling the expression of a nearby gene or gene cluster) could impart new instructions on the transgene. Consequently, it is essential that several independent lines of mice, derived from founders with different insertion sites, are examined before a conclusion relating a phenotype to a specific pattern of transgene expression is made.
When performing gene targeting in embryonic stem (ES) cells, position effects are essentially eliminated but not the effects caused by genetic variability. As in the transgenic experiments, this results from the generation of hybrid strains. Most commonly used ES cell lines are derived from strain 129, and a number of 129 substrains are in existence (129/Sv, 129/SvEv, and 129/Ola), further complicating the scenario. As mentioned above, hybrid vigor has been reported for the viability of ES cell lines.22 Although many (but not all) ES cell lines are themselves inbred, most investigators report that the 129 strain exhibits poorer reproductive performance than other inbred strains and also exhibits other abnormalities, including development of teratocarcinoma. This has prompted most investigators to breed their chimeras to C57BL/6, thus generating a hybrid mouse that is heterozygous (+/-) at the target locus and an F1 between C57BL/6x129 at all other loci. The F1 mice are all genetically identical because they inherit 1 chromosomal complement each from the 129 and C57BL/6 strains. However, when they are intercrossed to generate a mouse homozygous (-/-) for the target locus, the resultant offspring become an F2 of the parent strains. Therefore, whether wild-type, heterozygous, or homozygous for the target locus, the offspring have a random mix of 129 or C57BL/6 chromosomal DNA throughout the genome. The maintenance of a strain homozygous at the target locus by continuous inbreeding of these F2 mice can eventually select for phenotypic changes because loci causing deleterious effects are lost, and those providing a survival advantage are retained. Consequently, maintenance of the targeted locus in this manner is not recommended. Therefore, investigators are again left with the option to retain the mixed genetic background of the strains or to generate congenic strains.
Further complicating this problem has been the marked increase in the generation of double-knockout strains and the combinatorial use of knockout and transgenic rescue. In the latter, transgenes expressed either systemically or tissue-specifically are transferred into a knockout mouse to rescue some altered phenotype (often lethality).23 Moreover, the development of inducible transgenes and methods using the cre-loxP recombinase system to generate cell-specific knockouts will necessitate the introduction of multiple transgenes into a single genetic background. Clearly, it will become important to avoid the creation of a mixed genetic background so complex as to preclude any reasonable use of controls and prevent replication by other investigators.
| Effective Experimental Strategies |
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Although the generation of congenic mice is simple, requiring only accurate record keeping, it can be time-consuming and expensive, especially when multiple lines must be developed. Six generations of backcross breeding (2 years) is required before the genetic backgrounds are statistically >99% homogeneous, and the return on additional generations of backcross breeding markedly diminishes thereafter. For example, it requires 4 additional generations to increase genetic homogeneity from 99.2% to 99.95%. The use of a speed-congenic approach or a combination of in vitro fertilization and prepubertal superovulation can be used to decrease the time needed to generate congenic strains.24 25 The speed-congenic approach makes use of the well-developed genetic map of the mouse, thus affording an opportunity to screen the DNA of each offspring generated along the route toward congenic production to select for mice containing markers from the appropriate genetic background at the target locus and elsewhere.26 27 Those mice that are "further" along in congenic development than expected, on the basis of random segregation alone, can be selected by this process for further breeding.
After a C57BL/6 congenic knockout strain is derived, either
nontransgenic littermates or age-matched inbred C57BL/6 mice should
serve as reasonable controls. However, it is important for the
researcher to appreciate that even this scenario has weaknesses.
Indeed, 1 limitation of using littermate or wild-type mice as controls
for congenic transgenic (or knockout) strains is that some parental
genomic DNA upstream and downstream from the target locus (129 DNA in
the case of ES cellderived gene targeting) remains. By use of the
same example as described above, this occurs because as the targeted
modification (made in 129 genomic DNA) is introgressed into the C57BL/6
strain, the targeted locus and therefore 129 DNA in the vicinity of (or
linked to) the locus will be selected in each backcross generation
(Figure 2A
). The location of the
breakpoint between C57BL/6 and 129 genomic DNA upstream and downstream
from the target locus will depend on where the recombination between
the 2 genomes occurred. Therefore, nontransgenic littermates and
wild-type C57BL6 mice will lack the targeted locus and also the closely
linked 129 genomic DNA (Figure 2B
). In some cases, this DNA may
contain closely linked modifier genes. This may be especially critical
when examining large gene families, which may have members closely
clustered in the genome.
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Therefore, the optimal control would be a congenic control strain
containing a similar amount of foreign genomic information around the
target locus but lacking the targeted modification itself (Figure 2C
). Absolutely identical congenic strains cannot be generated.
However, similarities between control and experimental strains can be
maximized by taking advantage of the dense genetic map of the mouse and
the thousands of polymorphic microsatellite markers distributed
throughout the mouse genome.28 For example, control mice
can be selected that contain C57BL/6-specific markers throughout the
genome except in the region of the target locus, where 129-specific
markers would be selected. These mice can then be propagated to
generate a control congenic strain that is similar to the experimental
mouse but lacks the targeted ES cellinduced modification.
Of course, we must recognize that from a practical standpoint, circumstances will often dictate assessing the phenotype of a knockout mouse long before a congenic strain can be generated. If these mice were derived from 129 ES cells and the chimera was bred to C57BL/6, it is predictable that each mouse, while containing the same targeted modification of the genome, will be genetically different at all other loci because of random segregation and recombination in the F2 generation. In this case, it would be inappropriate to use either inbred C57BL/6 or 129 mice solely as controls. Instead, wild-type or heterozygous littermates from the same breedings should be included as well. The use of littermates would help minimize environmental variability in such experiments. Moreover, larger numbers of mice should be examined to ensure that the range of phenotypes possible due to epigenetic interactions with the genetic background is observed. Although not genetically identical, when examined as a population, the experimental and control groups could be considered "genetically similar." Once the phenotypes are assessed, it would be prudent to generate congenic strains and reexamine the phenotype in the resultant animals.
| Guidelines |
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Minimally, these guidelines provide a common-sense approach that provides the reader with sufficient information to understand and potentially replicate reported results and also provide a framework to identify the causes of phenotypic variation observed in different laboratories. It makes common sense to recommend that these guidelines be adopted by all researchers using genetically modified mice as models of cardiovascular disease until such time as standardized strains are used universally.
| Footnotes |
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Received January 6, 2000; accepted February 28, 2000.
| References |
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