- The Jackson Laboratory

Congenic and Mixed Background Strains from the Induced Mutant Resource

Several of the transgenes or targeted mutations in the Induced Mutant Resource (IMR) are being placed on inbred strain genetic backgrounds, primarily the C57BL/6J (B6) background. All mutations or transgenes will be backcrossed to the host strain at least 10 generations. To make them available to investigators for research as rapidly as possible we distribute some strains (called incipient congenic strains) at generations less than N10.

NOTE: There is a statistical probability that mice from these incipient congenic strains at <N10 are still segregating for genes from both the host (usually B6) and donor (usually 129) genetic backgrounds. The region around the mutation or transgene will be like the donor (non-B6) strain. The backcross generation of mice you receive will be shown as the N number on the identification form attached to the top of each shipping container. Be sure to record this information. The lower the N number, the more genes you can expect to be segregating. Therefore, any variations seen in results may be due to differences in the genetic backgrounds of individual mice. If a mutation is distributed as homozygotes, mice of the host background (e.g. B6) are probably the best control, but be aware that mutants and controls may differ at genetic background loci, i.e. other than the mutant locus. The graph below shows the percent of the genome estimated to be of host (usually B6) origin at various backcross generations.

Sampling: If a mutation is provided as heterozygotes, each mouse has been verified to be a heterozygous carrier by a DNA test or protein assay. Tested mice will have the tip of the tail removed or have been subjected to retroorbital bleeding.

Coat color: Most targeted mutations are made in 129/Sv-derived embryonic stem cell lines. 129 sublines may carry white-bellied agouti (A^w), albino (Tyr^c) and chinchilla (Tyr^c-ch) alleles at the albino locus, or pink-eyed dilution (p). Thus, intercrosses of black (a/a) mice from either <N10 B6 congenic lines and/or mixed stocks can result in progeny with coat colors other than the expected nonagouti black of B6. Depending on the combination of alleles, coats of mice may be albino or show varying shades of lightened color. You may also receive mice of these colors or white-bellied agouti from stocks with mixed backgrounds or B6129F2 controls.

Immunological traits: Congenic strains at <N10 may not be usable for immunological experiments that require histocompatibility (e.g. transplantation studies) or a consistent genetic background. For example, mice may reject transplanted tissues from each other or B6 for reasons unrelated to the experimental protocol, because they may differ at minor histocompatibility genes. If you want to try immunological experiments in mice at <N10, you can test mice for transplantation efficiency. Be aware, however, that at generations <N10, individual mice from the same generation cannot be expected to be genetically identical.

Controls for Targeted Mutant Stocks

Most targeted gene knockouts have been made in embryonic stem (ES) cells derived from the 129 strain. The mutated ES cells are usually injected into C57BL/6 (B6) blastocysts. The resulting chimeras contain cells of C57BL/6 and 129 origin. Frequently the chimeras and their progeny are mated inter se (sibling matings), which results in the genetic background being a mixture of C57BL/6 and 129 genes. Below we describe how such stocks are designated and the appropriate controls we provide. Note also that the strain origin of ES cell lines used for targeting mutations may vary; again see below for more information.

Nomenclature

The term STOCK has traditionally been used in strain nomenclature at The Jackson Laboratory to designate inbred strain backgrounds that contain genes from more than two inbred strains or unknown sources, resulting in a "mixed" strain. We have extended use of the word STOCK to designate strains derived from chimeras, e.g. B6<-->129 . Initially these STOCKs may be segregating for alleles at any loci that differ between the two parental strains, e.g. B6 and 129. In other words, the genetic make up of mice from the STOCK will vary from generation to generation until it becomes inbred (brother x sister matings for 20 or more consecutive generations). Once the strain is inbred, all mice will be genetically identical with genes fixed at every locus and derived from one or the other parental strains.

Recent Changes

We have recently changed the genetic nomenclature for targeted mutation and transgenic strains to enhance the amount of information the symbols contain about genetic background. Because most targeted mutant mice are derived from two known strains (not necessarily always B6 and 129), we have changed the strain nomenclature to reflect this information. Strains previously designated STOCK will now be designated by abbreviations for the two strains of origin separated by a comma if such strains contain only genes from two inbred genetic backgrounds. e.g B6,129. A stock labeled B6,D2 is derived from only the C57BL/6 and DBA/2 genetic backgrounds. The new nomenclature distinguishes such mixed background strains from F1 hybrids derived from the same inbreds; the F1 hybrid designation lacks a comma between the strain abbreviations.

Controls

For stocks of mixed or segregating genetic background, littermate controls are best. Many of the stocks received by the Induced Mutant Resource (IMR), however, are maintained by homozygous sibling matings and, therefore, wild-type littermates are not available. Physiological controls for strains on a mixed C57BL/6 x 129 background are provided from one of two B6129F2/J colonies (Stock Numbers 100903 and 101045). Like the targeted mutant mice, the genetic background composition of B6129F2 mice varies among littermates because of gene segregation from the F1 hybrid parents. Although these F2 mice only provide an approximate genetic match to the STOCK or B6,129 background, like the STOCK background they only contain genes derived from the C57BL/6 or 129 genetic backgrounds. Note that the B6129F1 hybrid is a less appropriate control because all F1 mice are genotypically identical.

Origin of ES Cell Lines

Until recently, we provided as a control an F2 between the two inbred strains C57BL/6J-A^w-J and 129/J . We have now researched the relationship of the various 129 substrains to themselves and to the ES cells commonly used by investigators to produce their targeted mutants. It appears that many ES cell lines have been derived from a 129/Sv congenic strain that carried the steel (Mgf^Sl-J) mutation. This congenic was made by backcrossing the steel-J mutation from C3H/Hu 12-14 generations to 129/Sv (by Dr. Leroy Stevens). Thus, this strain (now designated 129/Sv+^p+^Tyr-c+^Mgf-SlJ, because we have removed the steel-J mutation from it) is most closely related to the ES cell-derived 129 substrains. Therefore, we are now using the 129/Sv+^p+^Tyr-c+^Mgf-SlJ subline mated to C57BL/6J-a/a to produce B6129F2/J controls. Note that these F2 mice will be wild type at the albino/chinchilla (c, c^ch), pink eye (p), and steel (Mgf^Sl-J) loci. They will segregate ONLY for a and A^w-J at the agouti locus. This F2 represents the best genetic control that The Jackson Laboratory can offer for B6,129 mixed stocks.

Two examples are given to show how one uses the genetic nomenclature information to determine the appropriate controls.

(1) The strain B6,SJL-TgN(WapHRAS)69Lln Y^SJL (Stock Number 2409) was imported on a B6,SJL mixed genetic background, making B6SJLF2 (Stock Number 100726) mice the best genetic control we can offer.
In contrast, the transgenic stock B6SJL-(SODG93A)1Gur^dl (Stock Number 2300) is maintained by mating hemizygous (one transgenic insertion) carriers to B6SJLF1 hybrids (Stock Number 100012) at each generation; so either B6SJLF1 hybrid mice or +/+ siblings are possible controls.

(2) B6,D2-TgN(MMTVTGFB1)46Hlm (Stock Number 2375) will be imported on a B6,D2 mixed background and we will offer B6D2F2 (Stock Number 100086) mice as controls.
B6D2-TgN(MMTVTGFA)29Rjc (Stock Number 2373) will be available on B6D2F1 (Stock Number 100006), therefore the B6D2F1 can be used as a control.

We will also provide mice from the 129/Sv+^p+^Tyr-c+^Mgf-SlJ/J (Stock Number 2448) strain itself as controls for targeted mutations that are maintained homozygous on a 129 genetic background because, once again, the mutations were often made in ES cells derived from this 129 genetic background. Depending on your experiments, it may be important to determine the 129 substrain used by the original investigator.

Often it is important to know the origin of the DNA (genes) immediately surrounding a targeted mutation. Not only are ES cell lines currently in use derived from different 129 substrains, but ES cell lines are now being made from strains other than 129. For example, three Il4 (interleukin-4) targeted mutations will be available shortly, made in ES cell lines derived from 129, BALB/c, and C57BL/6 -- C57BL/6J-Il4^tm1Cgn (Stock Number 2253), BALB/c-Il4^tm2Nnt (Stock Number 2496), and C57BL/6-Il4^tm1Nnt (Stock Number 2518).

If genetic background may affect your experiments, please refer to the original reference for strain background and ES cell origin information or contact Dr. Carol Linder, Technical Services Advisor (phone: 1-800-422-MICE ext. 4416 or 207-288-6230, e-mail: ccl@jax.org).

Stock #100903: progenitors C57BL/6J-A^w-J (this substrain is isogenic with C57BL/6J except at the agouti locus) and 129/J (Stock #000690) (Appropriate for targeted mutations made with ES cell lines derived from 129/J or 129/ReJ, i.e. E14TG2a).

Stock #101045: progenitors C57BL/6J (Stock #000664) and 129/Sv+^p+^Tyr-c+^Mgf-SlJ/J (Stock #2448) (Appropriate for ES cell lines derived from this 129/Sv congenic substrain, i.e. D3, J1, CCE, AB1, AB2.1; this 129 substrain is wild-type at the albino (Tyr^c) and pink-eye dilution (p) loci so that their coat color is white-bellied agouti rather than albino or light chinchilla).