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Paper review: Genetic variation among 129 substrains


"Genetic variation among 129 substrains and its importance for targeted mutagenesis in mice"; Elizabeth M. Simpson, Carol C. Linder, Evelyn E. Sargent, Muriel T. Davisson, Larry E. Mobraaten and John J. Sharp Nature Genetics Vol. 16, pp 19-27 (1997).

I believe that this is an important paper for all individuals involved in the generation of "knockout" mice, as the information contained within it allows the tailoring of the ES cells used by various institutions and groups to specific 129 substrains. This has several key advantages, which will be covered in this review.

There are benefits to having a clear understanding of the different substrains and the genetic background of ES cells to be used for targeted mutagenesis in mice.
  1. Marker alleles can be used advantageously
  2. The genomic library in which a targeting construct is prepared should match the ES cell into which it is introduced, as polymorphism may reduce the frequency of homologous recombination.
  3. A coisogenic inbred strain is possible only by crossing a 129-derived chimeric mouse to the same 129 substrain from which the ES cells for generating this chimera were themselves derived.
  4. If the targeted strain is homozygous, true genetically matched controls cannot be approximated without knowledge of the correct ES cell substrain.
In order to gain an appreciation and a sense of history as to how these substrains and ES cell lines were derived, it is essential to view the schematic genealogical tree. This small thumbnail picture may be clicked to access a much larger representation. Since this original strain (129) was originally established by Dunn, L.C. at Columbia University in 1928, there have been several changes in breeding strategies which have contributed to this strain's genetic diversity. Evidence for all 129 substrains not being the same is evident in the phenotypically obvious coat color alleles, and they have been developed using this marker over time. Indeed, the change in coat color in breeding colonies has oftentimes denoted times where breeding strategies have changed, by accident or design. There have been several interesting aspects to the history of the 129 strain, including colony loss by fire, re-establishment from former distribution sites, extinction of a production line by poor breeding performance and its re-establishment from another breeding colony. Perhaps the most significant event for those who use ES cells to generate "knockout" animals was the development of the substrain colony by Leroy C. Stevens, termed 129/Sv. His research effort was directed towards study of the genetic basis for testicular teratomas, for which males of the parental 129 line developed at a frequency of 1-3%. His research involved outcrosses to introduce mutations affecting the gonads or germs cells. It is this property of 129 substrains which is utilized in "knockout" mouse production - the ES cells from this particular substrain are more proliferative and likely to invade and take over development of the inner cell mass (ICM) of injected blastocysts. Leroy C. Stevens' work also led to the development of the "Steel" substrains by outcrossing to C3H/Hu carrying the steel-J allele at the mast cell growth factor locus. This mutation increased the teratoma incidence to 10%, and the mutant allele is kept segregating because the homozygotes are inviable. Some ES cell lines have been developed from these substrains also. Interestingly, there is another 129 substrain originating from Leroy Stevens' colony, originally termed 129/terSv. This was generated by an outcross to the (parental) 129/Sv to a WC x C57BL/6 hybrid to introduce a dominant spotting mutation (Kitw, formerly W). After 8 generations of backcrossing to 129/Sv, an increase in the testicular teratoma rate (to 30%) was observed. The Kitw allele was bred out of this colony, and this substrain formed the parental line to 2 subsequent colonies - one of which the Ter mutation was selected out. However, the other of these 2 colonies is (at the time of publication) only 10 generations removed from the original Stevens' colony. There is an interesting possibility that this colony or that of its predecessor (originally 129/TerSv, now 129/Sv-+p Tyrc-ch Ter/+), showing an increase in teratomas, might prove to be useful in generating an aggressive ES cell line.

This paper attempts to demonstrate the genetic disparity between the 129 substrains and some of the most commonly used ES cell lines. The methods of assessment used were the acceptance or rejection of skin grafts across substrains and the allelic variants of Simple Sequence Length Polymorphism (SSLP) markers.

Skin Grafts

This study observed a high extent and degree of skin graft rejection among the 129 substrains, which the authors attribute to the complex history of substrain development. This is not usually seen for the widely used inbred strains even after several generations of pedigree isolation. The observed graft rejections were ascertained to be due to minor histocompatability differences, as all substrains were classified as "b" at H2K and H2D (a major histocompatability complex associated with rapid rejection). Two groups of histocompatible substrains were identified by graft rejection analysis, generally the 129/J and pre-Stevens' substrains showed compatibility, and the Ter and Steel substrains were also generally compatible with each other. There were also incompatible substrains that rejected grafts from all other substrains and all other substrains rejected grafts from them; these were primarily the 129/Sv Stevens' derived substrains. It was concluded that the extensive rejections of parental substrains is uncharacteristic of genetic drift, and given the complex strain history was probably due to accidental outcrossing.

SSLP marker analysis

Allelic variants of 86 markers of 15 129 substrains and 10 ES cell lines were examined at an average 20 centiMorgan spacing (randomly chosen). 56 of these differed between 129 substrains and C57BL/6 (65%). 37 (43%) exhibited at least 1 polymorphism among the 129 substrains. Reported differences between substrains varied between 0 - 22 of loci tested. 129/Sv was excluded from these statistics as it is segregating at 14 loci; alleles from substrain 129/SvPas and ES cell lines R1, D3, EK, CCE and AB1 are also segregating. Reasons for SSLP polymorphisms include new mutations, breeding problems in congenic strain construction including the presence of linked genes or incomplete backcrossing, or breeding error. Using existing 129 substrains separated in 1948, the new mutation rate was determined, enabling the authors to make the following statement: "Our analysis indicates that, although mutations have occurred within the 129 substrains, the majority of the extensive variability is most likely the result of outcrossing or genetic contamination. Interestingly, based on SSLP data, there is no necessity to hypothesize an increased mutation rate in ES cells." Using the SSLP data, ES cell lines can be matched to a substrain to provide the best genetic match. There are only 3 ES cell lines which demonstrate a "perfect fit", maintaining the best environment for homologous recombination to occur when utilized with a matched genomic library. These are tabulated as follows:

ES cell line129 Substrain Jackson Labs Cat.#
RW-4*129/SvJ 000691
mEMS32**129/J000690
J1*** 129/SvJaeN/A

All other ES cell lines have some polymorphisms that may impact upon their rate of homologous recombination to some degree. Perhaps the counter-argument to this is the cell line R1, which is a hybrid between the parental and steel lineages. It is believed that it may owe its robust germline ability to its hybrid nature.

The recommendations that follow as a result of these analyses are as follows:

  1. Maintenance of a mutant on an inbred background allows more reproducibility in results obtained through a lack of variability and defined controls.
  2. Derivation of a homozygous mutant strain on a mixed genetic background will fix a new combination of alleles that may impact the targeted allele.
  3. When heterogeneity is a concern, backcrossing to the matched 129 substrain is recommended.
  4. Use of isogenic DNA for targeting to maximize homologous recombination (Deng, Capecchi, MCB 12, 3365-3371, 1992)
  5. Examine other factors in selecting the best 129 substrain for use, such as documented differences in reproductivity and behavior.


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