Читать книгу Pathology of Genetically Engineered and Other Mutant Mice - Группа авторов - Страница 55

A cross of two unrelated inbred mice (for example, A/J × BALB/cByJ) produces pups that are all identically 50% maternal genome and 50% paternal genome with no recombination between any of the chromosomes. This is called the first filial or F1 hybrid generation and is named by putting the maternal then paternal parental strain names in parentheses separated by a lower case x, to represent cross, and followed by F1, (A/J x BALB/cByJ)F1 (Figure 3.4). This can be abbreviated using the strain abbreviations (Table 3.5) and dropping the parentheses and x, ACByJF1 in this case (Figure 3.4). Aside from spontaneous mutations arising in that single generation, all pups from all litters of such a cross should be genetically identical and have hybrid vigor, which is increased fecundity and overall health resulting, in part, from the loss of homozygosity of detrimental recessive alleles. Additionally, F1 hybrid mice can generally accept tissue transplants from either parent (Table 3.6). Unlike inbred strains, F1 hybrid mice are not self‐perpetuating because at meiosis each germ cell will have a unique set of recombinations between each of the two parental chromosomes, except for the sex chromosomes outside of the pseudoautosomal region.

Figure 3.2 Finding the gene and allele symbol on Mouse Genome Informatics. One of the chapter authors inquired about how to find the correct allele symbol to use relative to a specific reference

(Source: Tamai Y, Nakajima R, Ishikawa T, Takaku K, Seldin MF, Taketo MM. Colonic hamartoma development by anomalous duplication in Cdx2 knockout mice. Cancer Res. 1999 Jun 15;59(12):2965‐70).

Go to the Mouse Genome Informatics home page (http://www.informatics.jax.org/) and select “Gene.” The link goes to “Genes, Genome Features & Maps.” Then select “Genes and Marker Query.” In the Gene/Marker query box, enter the gene symbol or mutant mouse name, in this case Cdx2 was entered. Note, while gene symbols should be in italics no italics are given on this website or many other websites. This will give you all the genes for which this is part of the symbol or where Cdx2 was used as a synonym at some point in time. Selecting Cdx2 will yield the “Gene Detail” page. In the lower right area is the link to find all 14 of the currently reported allelic mutations (as of 1 May 2020) or the number of specific types of mutations. By selecting “All Mutations and Alleles” a summary field will appear. But which one is correct? By looking at the last author on the reference it is obvious from the summary. This is confirmed by selecting “Cdx2^tm1Mmt” to reveal the data sheet on this specific allelic mutation. At the bottom of the field is the reference that is the original reference provided.

Source: The Jackson Laboratory.

Figure 3.3 Current symbols assigned to a variety of unrelated genes originally published as p38. If one searches for the gene symbol p38 the results indicate numerous genes on different chromosomes that at one time were all called p38. The new names not only separate out the different genes but also reflect to a degree what is currently known about the gene function. By following the links you can obtain much more detail on each gene, the number and details on allelic mutations, and links to the original and many subsequent publications on the topic (MGI accessed 22 April 2020).

Table 3.3 Inbred strains of mice.

AdvantagesGenetic and phenotypic uniformity (smaller numbers needed)Well characterized (pathology and physiology)For most standard inbred strains >200 generationsIdeal controls (both biological and sequencing)Permits clear genetic mappingEnables identification of modifier genes

DisadvantagesNot as robust (smaller, lower reproductive performance [fecundity], shorter lifespan)Strain‐specific characteristics (deleterious mutations causing strain specific diseases)Expensive (when difficult to maintain)

UsesWidely used in all types of researchModels for human diseaseBackground for mice with spontaneous and induced mutations

Table 3.4 Inbred strain nomenclature.

C57BL/6J or C57BL/10J

C57BL is the parental strain name (since C57BL/6J is the actual strain name)

6 or 10 indicate the substrain line number (C57BL/10J are prone to serious heart disease not seen in C57BL/6J) [8]

J indicates the breeder (The Jackson Laboratory) of these substrains

C3H/HeJ or C3H/HeN

C3H is the strain generated by Strong from a cross of Bagg albino with DBA

He is the substrain from the W.E. Heston laboratory at the National Cancer Institute

J indicates the subline received from Heston then bred by The Jackson Laboratory

N indicates the substrain bred at the National Institutes of Health

An inbred strain is designated in capital letters.

A substrain is identified using a forward slash (/) after the strain name followed by a lab code of the strain breeder.

Further substrains derived from the founder add to the end of the lab code of subsequent breeders without another forward slash.

Note that C3H/HeJ has a mutation in the Tlr4 gene making it highly susceptible to gram negative bacterial infection while the C3H/HeN substrain is wildtype for Tlr4.

Figure 3.4 Nomenclature for hybrid stocks.

Table 3.5 Examples of inbred strain abbreviations.

129P3/J = 129P3

129S1/SvImJ = 129S1

A/HeJ = AHe

A/J = A

AKR/J = AK

BALB/cByJ = CByJ

BALB/cJ = C

C57BL = B

C57BL/6J = B6

C57BL/6JEi = B6Ei

C57BL/6NJ = B6NJ

C57BL/10J = B10

C57BR/cdJ = BR

C57L = L

CBA/CaJ = CBACa

CBA/J = CBA

C3H/HeJ = C3

C3HeB/FeJ = C3Fe

DBA/1J = D1

DBA/2J = D2

NZB/BINJ = NZB

NZW/LacJ = NZW

RIIIS/J = R3

SJL/J = SJL or J

SWR/J = SW

Table 3.6 F1 and F2 hybrid mice.

F1 hybrids	F2 hybrids
AdvantagesGenetic and phenotypic uniformityContains a 50 : 50 mix of both parental strainsHybrid vigorAccepts transplants of tissue from mice of either parental strain	AdvantagesHybrid vigorF2 hybrids do not have 50 : 50 mix but do have some mice in the population homozygous for some alleles and are valuable as a population for mutant gene mapping
DisadvantageNot self‐perpetuating	DisadvantagesNot self‐perpetuatingGenotype and phenotype NOT uniformParental strains most likely reject tissue transplants
UsesRadiation researchBehavioral researchBioassays of nutrients, drugs, pathogens, or hormonesTransplant recipientsBackground for transgenics and some deleterious mutations	UseControl for many targeted (“knockout”) mutations on a mixed B6; 129

Sibling breeding F1 hybrid mice for one generation produces an F2 hybrid population (Figure 3.4), and continued inbreeding after that eventually leads to the creation of recombinant inbred strains (see below). The F2 hybrid population also has hybrid vigor, but is distinct from the F1 hybrid population because meiosis has resulted in recombinations between the parental chromosomes such that there may be some homozygosity in a minority of the segregating alleles. This makes an F2 hybrid population a tremendous tool for mapping, especially for mapping recessive mutations. For example, if an interesting phenotype is observed in an inbred strain, such as an abnormal hair coat, hair interior defect (hid) in all AKR/J mice, these animals can be crossed with another inbred strain, such as BALB/cByJ, that has normal hair (Figure 3.5). As hid in this example is an autosomal recessive mutation, the obligate heterozygous F1 progeny of this cross will all have a normal hair coat. When these heterozygous progeny are intercrossed to create F2 hybrids, 25% of these mice are expected to have the abnormal hair phenotype. The genetic interval carrying the mutant sequence derives from AKR/J so by screening the DNA from the mutant and unaffected mice in the population with molecular markers distributed evenly across all chromosomes, a pattern emerges in which markers specific to AKR/J and not BALB/cByJ skew to increased homozygosity in the region of the genome specific to the mutation. Usually this works relatively quickly to identify the region in which the mutated gene is located. However, in this particular case, less than 15% of the F2 mice were affected (Table 3.7) due to modifier genes in BALB/cByJ. Similar crosses with other strains resulted in the expected 25% distribution. This illustrates two points, the deviation from Mendelian expectation due to modifiers and the value of doing mapping crosses with different inbred strains, which in this instance identified different genetic intervals that together reduced the interval and the number of candidate genes (Figure 3.6) [9, 10].

Figure 3.5 Setting up a mapping cross using F1 and F2 hybrids.

Backcrossing an F1 hybrid to one of the parents, instead of sibling intercrossing, is the initial process in the creation of a congenic, consomic, or conplastic strain, and such an N2 population is a useful tool for mapping dominant mutations. Some mutations or transgenes are adequately deleterious to overall health that they cannot be maintained on an inbred background and instead require hybrid vigor. Such mutants are maintained by breeding to an F1 hybrid at each generation or through outcross–intercross to an F1 hybrid. One complicated example of this is B6EiC3Sn a/A‐Ts(17¹⁶)65Dn/J [11]. This chromosomal aberration was induced in DBA/2J inbred mice, but is detrimental to health and causes male sterility so in order to successfully maintain a colony a female carrier is bred to a (C57BL/6JEiJ x C3H/HeSnJ)F1/J male at each generation. There is no punctuation in the genetic background of the strain name, B6EiC3Sn, and the presence of the DBA/2J contribution is omitted from the nomenclature for simplicity and must be found in sources outside of the strain nomenclature.

Table 3.7 Breeding data used to identify the gene responsible for the hair interior defect phenotype.

AKR/J‐hid* homozygote crossed with	Total progeny	Normal mice	Affected mice	Affected (%)	X 2	p‐Value
BALB/cByJ	260	222	38	14.62	5.92	0.025–0.010
C57BL/6J	430	330	100	23.26	0.220	0.500–0.750
CAST/EiJ	339	255	84	24.78	0.003	>0.900
C3H/HeJ	248	191	57	22.98	0.170	0.500–0.750
FVB/NJ	272	205	67	24.63	0.006	>0.900
Total F2 mice	1549	1203	346

^* AKR/J‐hid was the original designation until the gene responsible was identified. Because the gene was identified with a different phenotype earlier, the allele is officially Soat1^ald not Soat1^hid.

Figure 3.6 Crossing AKR/J mice with more than one inbred strain to map the mutant gene locus. By using the shortest interval between different crosses, the list of candidate genes could be reduced. Sequencing then only had to be done on a few genes to identify the mutant gene responsible for the hair interior defect phenotype.

Sources: Based on Giehl KA et al. [9].

Some mutations are maintained on a background in which the genetic contributions of two inbred strains are segregating. The retarded hair growth (in ornithine aminotransferase, Oat^rhg) mutation arose spontaneously in AKR/J and was later outcrossed to C57BL/6JEi and subsequently maintained by sibling intercrossing [12, 13]. Thus, the genetic background consists of contributions from both AKR/J and C57BL/6JEi segregating at each generation. A semicolon between the strain abbreviations is used to represent this situation, B6Ei;AKR‐Oat^rhg/J. If more than two inbred strains contribute to the genetic background of a strain, then it is simply called a stock, such as STOCK Noto^tc/J (notochord homeobox gene, truncate mutation). The truncate mutation arose spontaneously in the F4 generation of a cross between a C3H female and a Swiss outbred male. It was subsequently bred to BALB/cHu and again to C3H before being inbred for more than 80 generations [14]. The STOCK is in capital letters designating inbred, and there is no hyphen between STOCK and the allele symbol. The strain could have been assigned a unique inbred strain name, such as TC/J, but was instead designated as a STOCK. When stock is in lower case, it indicates that the segregating population has not reached F20.