Germline Competence Research Articles

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 x 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 x BDF1 blastocyst injection. This Mol/MSM-1 ES line should provide an excellent new tool allowing the genetic manipulation of the MSM/Ms genome.

Molecular control of pluripotency and germ line competency in chicken embryonic stem cells

Molecular control of pluripotency and germ line competency in chicken embryonic stem cells

Fish ES Cells and Applications to Biotechnology

ES cells provide a promising tool for the generation of transgenic animals with site-directed mutations. When ES cells colonize germ cells in chimeras, transgenic animals with modified phenotypes are generated and used either for functional genomics studies or for improving productivity in commercial settings. Although the ES cell approach has been limited to mice, there is strong interest for developing the technology in fish. We describe the step-by-step procedure for developing ES cells in fish. Key aspects include avoiding cell differentiation, specific in vitro traits of pluripotency, and, most importantly, testing for production of chimeric animals as the main evidence of pluripotency. The entire process focuses on two model species, zebrafish and medaka, in which most work has been done. The achievements attained in these species, as well as their applicability to other commercial fish, are discussed. Because of the difficulties relating to germ line competence, mostly of long-term fish ES cells, alternative cell-based approaches such as primordial germ cells and nuclear transfer need to be considered. Although progress to date has been slow, there are promising achievements in homologous recombination and alternative avenues yet to be explored that can bring ES technology in fish to fruition.

Effective Production of Microinjectable Blastocysts for Germ-Line Transmission of Embryonic Stem Cells

Blastocysts of C57BL/6 mice obtained either 2.5 or 3.5 days post-coitum (dpc) were examined for efficient microinjection after overnight in vitro culture. Incidences of zona-free embryos were much higher at 3.5 dpc after natural mating (1.05/mouse) and superovulation (2.83/mouse) than at 2.5 dpc after natural mating (0.05/mouse) and superovulation (0.01/mouse). By testing germ-line competency of gene-targeted J1 embryonic stem cells, superovulation and/or in vitro culture should be recommended for producing microinjectable blastocysts for production of high germ-line chimeras.

Induction of primordial germ cells from murine epiblasts by synergistic action of BMP4 and BMP8B signaling pathways.

Extraembryonic ectoderm-derived factors instruct the pluripotent epiblast cells to develop toward a restricted primordial germ cell (PGC) fate during murine gastrulation. Genes encoding Bmp4 of the Dpp class and Bmp8b of the 60A class are expressed in the extraembryonic ectoderm and targeted mutation of either results in severe defects in PGC formation. It has been shown that heterodimers of DPP and 60A classes of bone morphogenetic proteins (BMPs) are more potent than each homodimers in bone and mesoderm induction in vitro, suggesting that BMP4 and BMP8B may form heterodimers to induce PGCs. To investigate how BMP4 and BMP8B interact and signal for PGC induction, we cocultured epiblasts of embryonic day 6.0--6.25 embryos with BMP4 and BMP8B proteins produced by COS cells. Our data show that BMP4 or BMP8B homodimers alone cannot induce PGCs whereas they can in combination, providing evidence that two BMP pathways are simultaneously required for the generation of a given cell type in mammals and also providing a prototype method for PGC induction in vitro. Furthermore, the PGC defects of Bmp8b mutants can be rescued by BMP8B homodimers whereas BMP4 homodimers cannot mitigate the PGC defects of Bmp4 null mutants, suggesting that BMP4 proteins are also required for epiblast cells to gain germ-line competency before the synergistic action of BMP4 and BMP8B.

Efficient site-directed dna integration into es and hematopoietic cells

DNA mediated gene transfer to hematopoietic cells has been hindered by low gene transfer efficiency. In an attempt to achieve efficient, stable integration of potentially large DNA vectors at a site which confers expression in a defined and a predictable manner, we are exploiting a Cre/lox based recombination strategy. As an initial test in hematopoietic cells, murine BaF3 cells were used to introduce a hygromycin resistant gene flanked by 34 bp lox sites (floxed) using a retrovirus. Subsequently, co-electroporation of a floxed green flourescent protein (GFP) construct and the cre recombinase plasmid should mediate integration through the lox sites replacing hygromycin with GFP. Site directed, stable integration as confirmed by FACS and Southern blot analysis occurred at a high frequency ranging from 1–7% in 5 different BaF3 clones, or some 100 fold higher than the efficiency for a control GFP vector lacking lox sites. To assess if the expression characteristics of an initial integration site can be reutilized to express a transgene in a defined and predictable manner, floxed GFP expressing BaF3 clones were chosen that had clonally distinct levels of GFP expression that spanned over 300 fold. This differential expression was maintained following cre mediated replacement with a floxed cDNA human Thy-1 construct. Analogous findings were also obtained in the human K562 cell line. As an initial effort to extend this approach to primitive primary cells, we introduced lox sites into murine embryonic stem cells (ES) cells either by a retrovirus or by a plasmid. Subsequent cre mediated integration again occurred with high efficiency—up to 22% and clones retained in vitro differentiation capacity and germline competency. Site directed DNA integration should provide a powerful new approach for studying gene function and regulatory mechanisms in hematopoietic cells.

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Easy assessment of ES cell clone potency for chimeric development and germ-line competency by an optimized aggregation method

Production of germ-line competent chimeric mice from embryonic stem (ES) cells is an inevitable step in establishing gene-manipulated mouse lineages. A common method used for creating chimeric mice is the injection of ES cells into the blastocoelic cavity (blastocyst injection). The aggregation method is an alternative way to introduce ES cells to the host embryo which is less difficult than blastocyst injection. Here we re-examined the condition of embryo-ES cell coculture on the aggregation method and found improvement of germ-line competent chimeric production by a simple modification of the coculture medium. Moreover, R1 ES cell and its 10 gene-manipulated subclones were tested by this method. Although all ES cell clones showed good morphology and a normal karyotype, the efficiency of chimeric development and germ-line transmission varied among clones and were classified into three grades according to germ-line competency. In the first group (class A), both the incidence of chimera with high ES cell contribution and the rate of germ-line transmission were fairly high. Germ-line competent chimeras were obtained but with rather low efficiency in the second group (class B), while another group (class C) showed an absence of high ES cell-contributed chimeras and no germ-line transmission. These results suggest the usefulness of this modified aggregation method to predict the potency of ES cell clones for germ-line competency.

A New Embryonic Stem Cell Line with Germ-line Competence in the FvB/N Background

A New Embryonic Stem Cell Line with Germ-line Competence in the FvB/N Background