The generation of genetically modified mouse strains by homologous recombination in embryonic stem (ES) cells is a major research tool in diverse fields of biology. Historically the majority of manipulations have been performed in ES cells derived from the 129 strain 
comprised of a wide range of genetically variable substrains not suitable for some studies, in particular immunology 
and neurobiology 
. Germline transmission breeding and subsequent backcrosses to C57BL/6 (B6) to generate congenic strains delay functional studies and increase costs, while at the same time genes closely linked to the original targeted locus remain from the original ES cell genome and may confound research results 
. Together, these considerations led the large scale mouse mutagenesis projects organized under the umbrella of the International Knockout Mouse Consortium (IKMC) to select B6-derived ES cells as the parental lines in which to mutate all protein coding genes of mouse 
. Improved methods for the generation of germline transmitting chimeric mice from B6 ES cell lines will make the IKMC and other B6 ES cell resources more accessible to the broader biomedical research community.
B6 ES cell lines have been available since the early 1990s 
; however, in spite of the clear advantages of the inbred background few targeted mutations have been published compared to the widely used 129 ES cell lines 
. B6 ES cells appear to be less efficient in generating germline transmitting chimeras 
. A recent report with a large number of clones showed only about 43% vs.
81% of germline transmitting clones for B6 vs.
129S5 ES cells 
. B6 ES cells have been shown to be more difficult to maintain in culture 
and more likely to become aneuploid suggesting instability and deterioration in standard culture conditions 
. Furthermore microarray analysis of gene expression patterns in cultured 129- and B6-derived ES cells showed that B6 ES cells have a greater tendency to lose their pluripotency in culture in standard 15% FBS LIF-supplemented ES cell medium than 129 ES cells 
Various methods have been employed to address the apparent specific requirements of B6 ES cells and to improve methods for their derivation and culture. The use of RESGRO™ medium (Millipore) almost doubled the efficiency of B6 ES cell derivation and generation of germline chimeras and completely ES cell-derived animals by aggregation with tetraploid embryos 
. The use of optimized KnockOut™ DMEM medium and chemically defined KnockOut™ Serum Replacement (KOSR; Invitrogen) also facilitated the derivation of karyotypically stable and germline competent B6 ES cells 
. The addition of various signal transduction pathway inducers and inhibitors have resulted in further improvements in ES cell culture and chimera production in various mouse strains including those previously thought to be non-permissive 
. It is currently postulated that this system of inhibitors constitutes a generic culture condition for the maintenance of authentic pluripotency 
. Here, we describe a KOSR-based medium for the efficient generation of germline chimeras using our B6-derived ES cell line, designated C2, and its targeted subclones.
A variety of host embryos and methods have been used to generate germline chimeras from B6 ES cells such as BALB/c and C57BL/6-Tyrc-2J
/J (albino B6/J) blastocyst injection 
and outbred CD-1 
and Swiss Webster (SW) 
morula aggregation. Laser-assisted microinjection into 8-cell embryos has yielded completely ES cell-derived animals 
. Efficient germline transmission from IKMC targeted B6 ES cell clones was demonstrated recently by Pettitt and colleagues 
blastocyst injections. However, C57BL/6-Tyrc-Brd
animals are not easy to obtain; similar C57BL/6-Tyrc-2J
mice available from The Jackson Laboratory have a relatively high cost and often require the maintenance of an in-house breeding colony; finally, the classical BALB/c host strain is not very efficient for producing a good number of quality embryos. For these reasons, alternative host embryos for B6 ES cells have been investigated, e.g.
Aggregation of cleavage stage embryos with ES cells 
offers an accessible method for generating ES cell chimeric mice. Since the early 1990s we have exclusively used the aggregation method to generate multiple genetically modified mouse strains from 129S1×129X1 (R1) 
and 129S6B6F1 (G4) 
ES cells. Here, we show that ICR morula aggregation is also an efficient method for producing germline transmitting chimeras from B6N ES cell lines. Taken together, the improved culture medium and a more accessible technique for generating chimeras will improve accessibility of the IKMC and other B6 ES cell resources to the broader biomedical research community.