ES Cells Used by the UMMS TAMC for Gene Targeting

 Genetic manipulation of the mouse genome has been customarily performed using ES cells derived from the agouti 129-inbred strain of mouse. This is because 129-derived ES cells have proven to be very robust in generating chimeras, with the agouti (129-derived) coloration easily visible on the black (C57BL/6-derived) coat in the chimeras. Furthermore, 129-strain ES cells often contribute to the germline of the chimeras, and when breeding chimeras with C57BL/6 test mice, the dominant agouti color in offspring signifying germline transmission is also easily detected in the G1 generation litters. However, the resulting targeted G1 mice are of a mixed 129 x C57BL/6 genetic background. Depending upon the study, these mice often have to be backcrossed 9 more generations with C57BL/6 mice to place the mutant allele on an inbred C57BL/6 background. The UMMS TAMC has routinely utilized a subclone (MK6) of 129SVevBrd derived ES cells in gene-targeting experiments. In addition, we have on hand AB2.2 (Hprt-deficient) ES cell, as well as several other versions of 129-based ES cells.

Recently, researchers from the Sanger Institute (UK) have isolated stable and germline competent embryonic stem (ES) cells from C57BL/6N mice (1). These ES cells allow for the generation of gene‐targeted mice on a C57BL/6 genetic background without the need for multiple backcrossing of mice. In addition, these ES cells are the foundation for two large-scale knockout mouse programs designed to provide targeted BL/6 ES cells to the scientific community (EUCOMM and KOMP). To generate suitable C57BL/6 embryonic stem cells for gene targeting experiments, the Sanger team established a male cell line (JM8) from the N-substrain of C57BL/6 mice. When these C57BL/6N-based JM8 cells were injected into blastocysts from albino mice, a high proportion of chimeras displayed ES cell contribution to both germline and somatic tissues. Two further sub-lines were then established: JM8.F6 (feeder-dependent) and JM8.N4 (feeder-free), that were tested for their performance in high-throughput gene targeting experiments. In these experiments the germline transmission rate exceeded 65% for multiple tested clones. Experience has shown that injection of black C57BL/6N embryonic stem cells into albino C57BL/6JTyrc-Brd blastocysts is a particularly favorable combination for germline transmission. However, a disadvantage of this combination is that a slightly mixed C57BL/6 genetic background (C57BL/6N x C57BL/6J) is produced when breeding the resulting chimeras with C57BL/6JTyrc-Brd albino mice to test for germline transmission in the G1 generation. Of course, one could breed the chimeras with inbred C57BL/6N mice to maintain a pure C57BL/6N substrain background- but then one could not use coat color in the G1 generation to identify germline-transmitting chimeras, as all the offspring would be of a black coat color regardless of germline transmission. Thus, the Sanger team decided to repair the non-agouti mutation in the JM8 cells.

The non-agouti mutation in C57BL/6 strains is due to an 11.8 Kbp retrotransposon located in the first intron of the agouti gene that abolishes the expression of the Agouti gene. The Sanger group performed gene targeting in the JM8 ES cells to delete the retrotransposon from the agouti locus and restore agouti gene function, permitting the visualization of ES cell-derived mice on an inbred C57BL/6 background by agouti coat color. One of the 'restored' clones (JM8A3) showed particularly favorable results in blast injection experiments. Since these JM8A3 cells are heterozygous for the corrected agouti allele [Atm1brd], crossing the resulting brown-on-white chimeras with C57BL/6N test mice yield embryonic stem cell–derived offspring with either agouti or black coats. A germline transmission rate of 80% was reported from the injection of targeted JM8A3 clones (2). As expected, half of the agouti offspring from JM8A3-based chimeras will have the targeted allele of interest. However, one still has to genotype all of the G1 generation mice from germline transmitting chimeras or risk losing some black colored mice that are ES cells derived. But you can easily identify the germline transmitting chimeras by the presence of agouti coat colors in the G1 generation, and performing tail biopsies and genotyping any offspring from that proven chimera will subsequently identify germ-line transmitted mice. This approach allows you to maintain the inbred C57BL/6N status of the offspring.

The UMMS Transgenic Animal Modeling Core (TAMC) also has JM8-strain ES cells for targeting, and routinely injects either JM8.F6-strain ES cells or JM8.A3-strain ES cells into albino C57BL/6JTyrc-Brd blastocysts. The resulting chimeras are either black (F6-derived) or agouti (A3-derived) on white. Since there is very little difference in the genetics (only 102 of 139,561 genotyped SNPs are discordinant between the C57BL/6N and C57BL/6J sub-strains) and since few differences exist in the behavior between the BL/6N and BL/6J sub-strains of C57BL/6 mice (2, 3), many UMMS Investigators choose to breed their TAMC chimeras to the commercially-available, albino C57BL/6JTyrc-Brd mice (4). Any resulting black or agouti F1 generation mice are tail-biopsied and genotyped, and half of these dark coat colored F1 mice will have the targeted allele of interest. Alternatively, the investigator may choose to breed the chimeras obtained from the TAMC to pure inbred C57Bl/6J or C57BL/6N mice (5) - but please note that all of the resulting mice should be genotyped for transmission of the targeted allele.

References/notes:

(1)    Pettit S J et al. 2009. Agouti C57BL/6N embryonic stem cells, a foundation for mouse genetic resources. Nat. Methods 6:493-5.
(2)    Mekada K, et al. (2009) Genetic differences among C57BL/6 substrains.. Exp Anim. 58(2):141-149.
(3)    Bryant CD, et al (2008) Behavioral differences among C57BL/6 substrains: implications for transgenic and knockout studies. J Neurogenet. 22(4):315-31.
(4)    C57BL/6J-Tyr[c-2J]/J (albino-B6) mice and C57BL/6J mice are available from the Jackson Labs: cat numbers 000058 and 000664, respectively.
(5)    C57BL/6N mice are available from Taconic: cat number B6.

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