TECHNOLOGY REPORT Improved Generation of C57BL/6J Mouse Embryonic Stem Cells in a Defined Serum-Free Media Jun Cheng, Amalia Dutra, Aya Takesono, Lisa Garrett-Beal,* and Pamela L. Schwartzberg* Genetic Diseases Research Branch, National Human Genome Research Institute, Bethesda, Maryland Received 10 December 2003; Accepted 25 February 2004 Summary: C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurolog- ical research. The establishment of C57BL/6 ES cell lines has facilitated the study of gene-altered mice in a pure genetic background— however, relatively few such lines exist. Using a defined media supplement, knockout se- rum replacement (KSR) with knockout DMEM (KSR-KD- MEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were es- tablished, all of which were karyotypically normal and could be maintained in the undifferentiated state on mouse embryonic fibroblast (MEF) feeders. One line was further tested and found to be karyotypically stable and germline competent, both prior to manipulation and af- ter gene targeting. For this cell line, efficiencies of cell cloning and chimera generation were greater when maintained in KSR-KDMEM. Our work suggests that the use of defined serum-free media may facilitate the gen- eration of ES cells from inbred mouse strains. genesis 39:100 –104, 2004. Published 2004 Wiley-Liss, Inc. † Key words: embryonic stem cells; gene targeting; defined media; KSR-KDMEM; C57BL/6J Embryonic stem (ES) cells have provided a powerful tool for the study of gene function and regulation, the gen- eration of animal models for human diseases, and the investigation of cell differentiation (Kokron et al., 1997; Shastry, 1998). Most available ES cell lines are derived from various 129 mouse strains. However, given the wide range of 129 substrains and the genetic variability of these strains, many researchers backcross gene-tar- geted mice to other inbred strains. For a number of reasons, the C57BL/6J mouse strain has been extensively used as a reference strain (Kontgen et al., 1993). The phenotypes of many mouse mutants have been studied in the C57BL/6J strain, including a variety of immuno- logical and behavioral mutants (Crawley et al., 1997). Second, because the genome of C57BL/6J is being se- quenced as part of the Human Genome Project, exten- sive mapping, sequence, and genetic data are available for this strain (Nadeau et al., 2001). Finally, generation of conventional transgenic mice in 129 substrains has proved difficult, whereas numerous groups have gener- ated transgenic mice in C57BL/6. For all of these reasons, investigators have backcrossed mutant mice to C57BL/6, a laborious process that requires extensive backcrossing even with the use of speed congenics. Establishment of ES cell lines derived from C57BL/6J strain has been a major step to help facilitate the direct genetic alteration of mice in a pure C57BL/6J genetic background (Auerbach et al., 2000; Kawase et al., 1994; Kontgen et al., 1993; Ledermann and Burki, 1991; Lem- ckert et al., 1997). However, there are relatively few ES cell lines derived from C57BL/6J mice. In an effort to facilitate the establishment of ES cells lines, we utilized a defined media supplement, KSR, with an optimized KD- MEM. We have found that cells cultured in this media are more readily disrupted upon trypsinization, a critical step in the early culture of ES cells. Twenty-three blastocysts flushed from uterine horns of naturally mated 3.5-day pregnant C57BL/6J females were transferred onto MEF feeder layers and were left undisturbed for 6 days without passage. Of these, 18 blastocysts attached and 15 inner cell masses (ICM) grew. From these cell masses, six cell lines, each derived from an independent blastocyst, were established. The morphology of one such ES cell line is shown in Figure 1a. In contrast, using fetal bovine serum-DMEM (FBS- DMEM), our usual growth media for ES cells, we were unable to obtain any cell lines from 19 blastocysts cul- tured in the same experiment. A repeat experiment gave similar results with three ES cell lines derived from 24 C57BL/6J blastocysts cultured in KSR-KDMEM. In a total of five experiments, we cultured 105 C57BL/6J blasto- cysts in FBS-DMEM from which we were able to see 32 ICMs. However, in our hands we were unable to estab- lish any ES cell lines from these cultures (Table 1). * Correspondence to: Lisa Garrett-Beal or Pamela L. Schwartzberg, Na- tional Human Genome Research Institute, 49 Convent Drive, National Institutes of Health, Bethesda, MD 20892. E-mail: lgarrett@mail.nih.gov or pams@nhgri.nih.gov † This article is a US government work and, as such, is in the public domain in the United States of America. Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/gene.20031 Published 2004 Wiley-Liss, Inc. genesis 39:100 –104 (2004)