Читать книгу Pathology of Genetically Engineered and Other Mutant Mice - Группа авторов - Страница 58

When a genetic mutation arises spontaneously or is created in a specific mouse strain and the investigator wants to characterize it on another inbred strain, this can be accomplished by a modification of inbreeding to create what is called a congenic strain (Table 3.9). For example, if you have an albino (white) strain carrying a single gene mutation, specifically a recessive mutation such as hairless on the HRS/J inbred strain (donor strain), and want to move it onto a predominantly C57BL/6J background (host strain), one parent from each strain is mated together to create F1 hybrids (see below). These F1 progeny are intercrossed to create F2 mice, ideally but not always 25% of which will be homozygous for the recessive hairless mutant allele. Only the mutant mice are then crossed back (backcrossed) to C57BL/6J mice to produce the N2 generation. This is repeated 10 times (N10) to create a fully congenic mouse strain [22, 23], with congenic nomenclature permitted to be used at N5. At N10, the strain will be mathematically >99% host background. Unfortunately, there will always be some of the parental strain DNA flanking the gene selected for, known as the congenic interval, and the possibility of trace amounts of donor sequence elsewhere in the genome. The more backcrosses to B6, the smaller this congenic interval should become and the lower the likelihood of traces of donor sequence but the genetic background will never entirely become B6. Creating a group of congenic strains on the same host background controls for phenotypic variability caused by genetic background in comparative or combinatorial assessments of mutation‐induced phenotypes.

This description is only true for recessive mutations that cannot be genotyped and even in those instances, a handful of researchers might go straight to N2 before the intercross, especially if it is a homozygote that cannot be used to breed. Dominant mutations or alleles that can be genotyped do not require the intercross step so can be repeatedly backcrossed at each generation.

Figure 3.7 Using recombinant inbred lines to narrow candidate gene intervals usingWWW.GeneNetwork.org The BXD lines on the left are all homozygous for the hypomorphic Abcc6 allele from DBA/2J. One quantitative trait locus interval has a mixture of DBA/2J (blue) and C57BL/6J (red) DNA. By phenotyping the mice, it is possible to narrow the interval to identify the modifier gene.

Source: Generated by Dr. Jason Bubier 4 May 2020; WWW.GeneNetwork.org.

Nomenclature for congenic strains begins with a standard abbreviation for the host strain (Table 3.4), followed by a period (.) to indicate congenicity, then the standard abbreviation for the donor strain, then a hyphen, then the allele(s) or congenic interval transferred, then a forward slash (/), and then laboratory code of the investigator in whose laboratory the congenic was completed. For example, a mutation in the sodium channel, voltage‐gated, type VIII, alpha gene (Scn8a^8J) was induced using N‐ethyl‐N‐nitrosourea (ENU) in the C57BL/6J background and was backcrossed onto C3HeB/FeJ in the laboratory of Dr. Wayne Frankel so the strain name is C3Fe.B6‐Scn8a^8J/Frk [24]. In the strain B6.C3‐Pde6b^rd1 Hps4^le/J both the phosphodiesterase 6B, cGMP, rod receptor, β‐polypeptide retinal degeneration one allele (Pde6b^rd1) and HPS4, biogenesis of lysosomal organelles complex 3 subunit 2 light‐ear allele (Hps4^le) were transferred in a single congenic interval from Chromosome 5 of a mutant subline of C3H/HeJ onto C57BL/6J. The congenic interval can also be represented not by the allele(s) of interest but by the most proximal and distal markers identified in the donor interval, such as in the strain B6.129S1‐(rs13480546‐rs13480629)/Kjn [25]. If two alleles of interest from separate genetic origins are combined into one congenic strain or if the precise origins of the donor sequence are not known, then there is no single donor strain that can be put after the period so that complex genomic donor source is represented as “Cg.” The Tg(Ins2‐rtTA)2Doi transgenic mice were generated by microinjection into (C57BL/6J x SJL/J)F1 hybrid mice then backcrossed onto a NOD host to generate the congenic named NOD.Cg‐Tg(Ins2‐rtTA)2Doi/Doi [26], in which the Cg is used because it is unknown whether the congenic interval derives from C57BL/6J or SJL/J. If the source of the desired donor sequence does not come from a strain that is a pure inbred, but rather contains contributions from a source different from the host background or the donor congenic interval, then that strain information can be represented in parentheses after the donor strain abbreviation. Thus, if it were known that Tg(Ins2‐rtTA)2Doi inserted into C57BL/6J‐derived sequence in the original F1 hybrid, and not into SJL/J‐derived sequence, then the strain name of the congenic would be NOD.B6(SJL)‐Tg(Ins2‐rtTA)2Doi/Doi. This allows the reader to see at a glance that there is a possibility that some small amounts of SJL/J‐derived sequence exist in the strain. In the example of NOD.129S4(B6)‐Art2a^tm1Fkn Art2b^tm1Fkn/Lt it is immediately clear that both targeted alleles were generated in 129S4, at least one, if not both, were bred at least once to C57BL/6J, and then both were backcrossed onto NOD, either together or separately then bred together [27]. If there are more than one additional sources of trace contribution to the genetic background, aside from the donor, then Cg is entered into the parentheses to represent this. For example, NOD.129P2(Cg)‐Il10^tm1Cgn Il4^tm1Cgn/Dvs had C57BL/6J and C57BL/10J in part of the genetic lineage so the potential contributions from those backgrounds are captured in the (Cg) [28]. Another example of a congenic strain in which the donor origin is represented by “Cg” is B6.Cg‐Kitl^Sl Krt71^Ca/J, a C57BL/6J host congenic for both Kitl^Sl, which arose in C3H/He, and Krt71^Ca, which arose in a Swiss stock [29].

Table 3.9 Congenic strains.

AdvantagesGenetic and phenotypic uniformityReduce experimental variabilityCan transfer most mutations onto a different genetic backgroundAllows examination of modifier genesCan maintain mutation or transgene homozygously and use inbred as control

DisadvantagesGeneration time (two to three years to N10 or longer if ovarian graft to immunodeficient mouse used; can reduce time with speed congenics)Phenotype may change on different genetic backgroundLinked genes may confuse experimental findings

As illustrated in the above examples, more than one gene can be selected for in the congenic breeding process, especially if molecular markers are available so that each generation can be screened. The entire process is speeded up if markers for the host strain are identified on all chromosomes and selected for during the inbreeding process, to create what are termed speed congenics.

The advantage of creating congenic strains is that the gene of interest is now on the inbred strain the investigator wants to work with. The disadvantage is that there is always some remaining DNA from the donor strain around the gene that has been moved and potentially elsewhere in the genome. The more the strain is backcrossed to the new strain, the smaller this interval is, but some donor DNA still remains, and may be in small segments of forced heterozygosity not linked to the congenic interval. Endonuclease‐mediated technology can be used to replicate the same allele sequence in different inbred strains, which eliminates the problem of flanking donor sequence in the congenic interval and saves years of breeding.

There are also a group of strains generated by the outcross of a coisogenic mutant subline followed by backcrossing to the original inbred strain in order to return to something close the original genetic background. This can occur by error or by necessity, such as when the chimeric founder of a targeted mutation created in an ES cell derived from 129S6/SvEvTac is bred to C57BL/6NTac to select mice with an agouti coat color, indicative of transmission from 129S6. If it is desirable to assess the phenotype of the allele on a 129S6 background, then repeated backcrossing to that original background can produce something similar to a congenic in that it is predominantly 129S6 but may carry traces of C57BL/6NTac sequence even though the interval surrounding the mutation is 129S6. Such a strain would be named 129S6(B6NTac)‐Gene^tm1Lab/Lab with the abbreviation of the predominant strain followed by the abbreviation of the potentially contaminating strain in parentheses. This nomenclature says at a glance that this is not a congenic strain (no period), the allele did not originate in C57BL/6NTac, but rather in 129S6‐derived sequence, but that some contribution from C57BL/6NTac may be present in the genome. This nomenclature is only used after five backcross generations. Prior to that the correct strain nomenclature would be 129S6;B6NTac‐Gene^tm1Lab/Lab.