Generation of C57BL/6 knockout mice using C3H x BALB/c blastocysts

The International Mouse Knockout Consortium aims to generate a knockout mouse for every single gene on a C57BL/6 background. Our ability to generate such mice is hampered by the poor economics of producing blastocysts to achieve germline transmission of C57BL/6 embryonic stem (ES) cells. We demonstrate superior utility of (C3H x BALB/c)F1 blastocysts compared with BALB/c blastocysts, with blastocyst numbers and germline transmission from subsequent chimeras at a rate 2- to 3-fold higher than that produced with BALB/c blastocysts.     

Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene function in vivo and differentiation into specific types as potential therapeutic agents for several human diseases. We describe here, the production of new ES cell lines from blastocysts recovered from the C57BL/6 and BALB/c mouse strains by changing the concentration of leukemia inhibitory factor (LIF) and primary culture conditions. The established cell lines were analyzed by simple karyotype, C banding, alkaline phosphatase activity, and Oct-4 expression as well as for the presence of the SRY gene. Two ES cell lines from C57BL/6 and three from the BALB/c were produced. The two C57BL/6 ES cell lines were established with either 1000 or 5000 IU LIF, whereas the BALB/c ES cell lines required 5000 IU LIF. Four of the ES cell lines had a normal karyotype. C banding and sex-determining region of Y chromosome-polymerase chain reaction showed that all cell lines had an XY sex chromosome composition. All five of the cell lines expressed alkaline phosphatase activity and Oct-4. One of the BALB/c ES cell lines, when injected into C57BL/6 blastocysts, produced high rates of chimerism as assessed by coat color, and the male chimera produced germ-line offspring when mated with BALB/c females. These results indicate that ES cells from inbred strains can be isolated using commercially available reagents and that the establishment of BALB/c ES cell lines may require different culture conditions to the 129 or C57BL/6 strains.     

Embryonic stem cells derived from C57BL/6J and C57BL/6N mice

Mouse embryonic stem (ES) cells with the C57BL/6 genetic background allow the generation of knockout mice without the need to backcross to C57BL/6. However, C57BL/6 ES cells whose pluripotency after homologous recombination has been confirmed are not yet available from public cell banks. To facilitate the use of ES cells derived from C57BL/6 sublines in both biologic and medical research, we demonstrated that the use of knockout serum replacement as a medium supplement and 8-cell blastomeres as recipient embryos allowed establishment of ES cells and production of germline chimeric mice, respectively. Under effective conditions, a large number of ES cell lines were established from C57BL/6J and C57BL/6N blastocysts. The majority of ES cells in many cell lines obtained from both strains showed a normal chromosome number. Germline chimeric mice were generated from C57BL/6J and C57BL/6N ES cells. Finally, the ES cell line B6J-S1UTR, derived from C57BL/6J, was used for successful production of gene knockout mice. C57BL/6J ES (B6J-S1UTR and B6J-23UTR) and C57BL/6N ES (B6N-22UTR) cells are available from the cell bank of the BioResource Center at RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

品系对小鼠胚胎干细胞分离效率的影响

为了充分利用小鼠胚胎干(ES)细胞,就必须从众多小鼠品系中分离ES细胞系。本研究通过传统的成纤维细胞饲养层法,从CD-1、129/Sv、C57BL/6J和129/Sv×C57BL/6J四种不同遗传背景的小鼠中分离得到12个ES细胞系,而从KM小鼠没有得到ES细胞系。所有的ES细胞系都具有典型的ES细胞特征,AKP染色呈阳性。从四种不同遗传背景的ES细胞系得到了包含多种组织的畸胎瘤;与桑椹胚聚合后,都得到了生殖系嵌合体。结果表明:品系对小鼠ES细胞的分离有显著影响,利用129小鼠以及包含129小鼠遗传背景的杂交小鼠都较容易分离ES细胞,由ES细胞得到生殖系嵌合体的效率在不同品系间有显著差异,从杂交ES细胞比近交ES细胞中更容易得到生殖系嵌合体。     

Gene targeting in C57BL/6 ES cells. Successful germ line transmission using recipient BALB/c blastocysts developmentally matured in vitro.

There are significant advantages to the production of gene-knockout mice directly in mouse strains other than 129. The availability now of ES cells derived from the C57BL/6 mouse strain presents workers with a valuable alternative. A major difficulty, however, is the requirement for BALB/c blastocysts as recipients for ES cell injection. Using standard procedures, few BALB/c blastocysts can be obtained. This limitation has now been resolved by harvesting BALB/c embryos at the early morula stage and maturing these to blastocysts by in vitro culture. Of early morulae harvested and cultured, over 70% were recovered as fully expanded and injectable blastocysts. C57BL/6 ES cell injection of these blastocysts has enabled the production of a number of gene-knockout mice with a success rate similar to that reported for ES cells derived from the 129 mouse strains.     

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

Transgenic mice ubiquitously expressing enhanced green fluorescent protein (EGFP) are useful as marker lines in chimera experiments. We established a new embryonic stem (ES) cell line (named B6G-2) from a C57BL/6 blastocyst showing ubiquitous EGFP expression. Undifferentiated B6G-2 cells showed strong green fluorescence and mRNAs of pluripotent marker genes. B6G-2 cells were transferred into a C57BL/6 blastocyst to generate a germline chimera, the progeny of which inherited ubiquitous EGFP expression. Mice derived completely from B6G-2 cells were also developed from the ES cells; these were tetraploid chimeras. The established B6G-2 cells were shown to be pluripotent and to be capable of differentiating into cells of all lineages. Thus, the new ES cell line expressing EGFP ubiquitously is useful for basic research in the field of regenerative medicine. The B6G-2 cell line is freely available from the BioResource Center, RIKEN Tsukuba Institute (http://www.brc.riken.jp/lab/cell/english/).     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

Yeast artificial chromosome targeting technology: an approach for the deletion of genes in the C57BL/6 mouse

An approach is described to modify yeast artificial chromosomes (YACs) with cassettes that can be easily excised for embryonic stem (ES) cell gene targeting experiments. YAC targeting technology (YTT) uses the WIBR/MIT-820 C57BL/6-mapped YAC library derived from the C57BL/6 mouse as the starting point for Internet- or PCR-based clone isolation, although in principle any YAC system can be used. Homologous recombination is initially performed in yeast using cassettes that function in Saccharomyces cerevisiae, Escherichia coli, and ES cells, followed by cloning or conversion of the targeted locus into a plasmid. The completed targeting vector can be transfected into C57BL/6 ES cells and clones selected with G418 followed by injection into Balb/c blastocysts. YTT increases the speed of targeting vector construction and obviates the need for extensive backcrossing to the C57BL/6 background.     

Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media

C57BL/6 is a well-characterized mouse strain that is used extensively for immunological and neurological 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 serum replacement (KSR) with knockout DMEM (KSR-KDMEM), we find that we can readily establish ES cell lines from blastocysts of C57BL/6J mice. Six lines were established, 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 after 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 generation of ES cells from inbred mouse strains.     

Establishment of germline-competent embryonic stem cell lines from the MSM/Ms strain

MSM/Ms is an inbred mouse strain established from the Japanese wild mouse, Mus musculus molossinus, which has been phylogenetically distinct from common laboratory mouse strains for about 1 million years. The nucleotide substitution rate between MSM/Ms and C57BL/6 is estimated to be 0.96%. MSM/Ms mice display unique characteristics not observed in the commonly used laboratory strains, including an extremely low incidence of tumor development, high locomotor activity, and resistance to high-fat-diet-induced diabetes. Thus, functional genomic analyses using MSM/Ms should provide a powerful tool for the identification of novel phenotypes and gene functions. We report here the derivation of germline-competent embryonic stem (ES) cell lines from MSM/Ms blastocysts, allowing genetic manipulation of the M. m. molossinus genome. Fifteen blastocysts were cultured in ES cell medium and three ES lines, Mol/MSM-1, -2, and -3, were established. They were tested for germline competency by aggregation with ICR morulae and germline chimeras were obtained from all three lines. We also injected Mol/MSM-1 ES cells into blastocysts of ICR or C57BL/6 × BDF1 mice and found that blastocyst injection resulted in a higher production rate of chimeric mice than did aggregation. Furthermore, Mol/MSM-1 subclones electroporated with a gene trap vector were also highly efficient at producing germline chimeras using C57BL/6 × BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.     

Culture of zygotes increases TRP53 [corrected] expression in B6 mouse embryos, which reduces embryo viability

The expression of TRP53 in blastocysts that had been cultured from the zygote stage in vitro for 90 h was compared with that in blastocysts collected from the uterus in C57BL6 (B6) and in F1 hybrid (B6CBF1) strain mice. In both strains, there was little TRP53 detected in blastocysts collected from the uterus. There was some increased expression in cultured embryos from B6CBF1 mice and marked increased expression in cultured B6 blastocysts. In cultured B6 embryos, there was obvious accumulation of TRP53 within the nuclear region of embryonic cells. Cultured B6 zygotes had significantly poorer rates of blastocyst formation and of capacity to undergo implantation or form viable fetuses than cultured zygotes from B6CBF1 mice or B6 blastocysts collected from the uterus. Trp53-/- zygotes (B6 background) were significantly more likely to form blastocysts than sibling wild-type embryos, with Trp53+/- embryos having an intermediate level of viability (P<0.01). On transfer of blastocysts to recipient females, Trp53-/- blastocysts were more likely to form viable fetuses than wild-type or heterozygous sibling blastocysts when the embryos resulted from culture of zygotes (P<0.001). This shift in viability did not occur when embryos were only subjected to 24 h of culture from the compacted embryo stage. Culture in vitro in the B6 strain caused a marked increase in the expression and nuclear accumulation of TRP53. This expression was a significant cause of the loss of viability that occurs on culture of zygotes from this strain in vitro.     

Increasing maternal age of blastocyst affects on efficient derivation and behavior of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) have the capability to undergo unlimited cell division and differentiate into derivatives of all three embryonic germ layers. These fundamental features enable mESCs to potentially be appropriate, efficient models for biological and medical research. Therefore, it is essential to produce high-performance mESCs. In the current study, we have produced mESCs from blastocysts that developed from fertilized oocytes of 2 (2-C57)-, 4 (4-C57)-, and 6 (6-C57)-month-old C57BL/6 mice. A comparison of isolated stem cells was done from the viewpoint of the efficiency of mESC derivation, self-renewal, and their differentiation capacity. All generated mESCs showed a similar expression of the molecular markers protein of pluripotency and AP activity. In the 3i medium, there was a significant decrease in undifferentiated marker genes expression in the 2-C57 cells compared with the other two groups ( P < 0.05) but developmental genes significantly increased in the 4-C57 and 6-C57 cells compared with the 2-C57 cells ( P < 0.05). The differentiation capacity into three germ layers through the embryoid body formation and percentage of cell lines with normal numbers of chromosomes reduced with increased maternal age. The highest DT and highest percentage of cells in the S phase belonged to 2-C57 cells. These data demonstrated that blastocysts which developed from fertilized oocytes of 2-, 4-, and 6-month-old C57BL/6 mice can generate pluripotent stem cells, and suggested that both the efficiency of mESC isolation and the behavior of these isolated mESCs including pluripotency, self-renewal, cell cycle, and DT changed with increasing maternal age.     

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.     

Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts

To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8?%) compared with B6NTac chimeric males (7/9, 78?%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86?%) B6J male chimeras were fertile compared with 6 of 11 (55?%) B6NTac male chimeras. Ten of 12 (83?%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50?%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42?±?1.73, n?=?12) compared to B6NTac chimeras (2.17?±?1.33, n?=?6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64?%) compared with chimeras produced using B6NTac blastocysts (4/11; 36?%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.     

Developmental responses of two substrains of in vitro fertilized C57BL/6J mouse embryos to oxygen and amino acids.

The developmental response of in vitro fertilized embryos to oxygen and amino acids were compared between in-house bred C57BL/6JNrs (Nrs) and commercially available C57BL/6JSlc (Slc) mice. Under 20% oxygen, the percentage of embryos that developed to blastocysts and expanded blastocysts, and nuclear numbers were lower in Slc embryos than in Nrs embryos. Moreover, the nuclear number did not increase in Slc embryos during 72-96 h culture. Effects of amino acids were beneficial on development of Slc embryos under 20% oxygen, but inhibitory on blastocoel formation at 78 h under 5% oxygen. On the other hand effects of amino acids on Nrs embryos were observed in nuclear number at 72 and 78 h culture under 5% oxygen. Because the present study showed differences in sensitivity to culture conditions between the C57BL/6J substrains, care must be taken in embryo manipulation of this inbred strain in studies of embryo development or other studies.     

Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture.

Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA. When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis.     

Establishment of a germ-line competent C57BL/6 embryonic stem cell line

Embryonic stem (ES) cell lines have been derived from blastocysts of the inbred mouse strain C57BL/6. The highest frequencies of ES cell colonies were observed when blastocysts were explanted directly onto growth-arrested feeder layers of 5637 human bladder carcinoma cells in the presence of conditioned medium. One of the male ES cell lines tested (BL/6-III) was shown to be karyotypically stable and germ-line competent when introduced into BALB/c host blastocysts. These results demonstrate that ES cell lines from inbred mouse strains other than 129/Sv may be used as vectors to introduce selected mutations into the germ-line of mice.     

Three inhibitors of FGF receptor,ERK, and GSK3 establishes germline‐competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability

C57BL/6 mouse is the most standard strain in mouse genetics. The strain does, however, have several disadvantages; one being the difficulty in establishing embryonic stem (ES) cells. No reliable C57BL/6 ES cell line is widely available for creating mutant mice through gene targeting. It also greatly favors mouse genetics if one can routinely make multiple mutations by stably culturing germline‐competent C57BL/6 ES cells or if one can routinely establish ES cells from C57BL/6‐derived mutant mice to make multiple mutations. Recently, an ES culture method with three inhibitors (3i: SU5402 for FGFR, PD184352 for ERK, and CHIR99021 for GSK3) has been reported. Here we show that this 3i method is extremely instrumental in establishing and culturing germline‐competent ES cells in the C57BL/6N strain. genesis 48:317–327, 2010. © 2010 Wiley‐Liss, Inc.     

Establishment of a novel embryonic stem cell line by a modified procedure

To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is diffucult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouseSry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.Abbreviations DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - FIAU 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil - LIF leukemia inhibitory factor - NEAA non-essential amino acids     

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.