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Jim McWhir

Publications and source records attributed to Jim McWhir.

13 recordsLinked to original sources
PubMed2004

Enhanced gene targeting frequency in ES cells with low genomic methylation levels.

Increased methylation in promoter/enhancer regions typically results in transcriptional downregulation. The direct correlation between gene expression and homologous recombination (HR) is also widely acknowledged, and suggests that actively transcribed, hypomethylated targets may be more accessible to the HR machinery. Consistent with this hypothesis, we report that DNA methyltransferase 1 (Dnmt1)-knockout ES cells show a 2-fold increase in gene targeting frequency. However, the use of hypomethylated targeting vectors or the ectopic expression of a putative DNA demethylase did not enhance targeting frequency. These observations are discussed in the context of devising more efficient targeting protocols by transiently modifying genomic methylation levels.

Animals↗
PubMed2003

Elevated expression of exogenous Rad51 leads to identical increases in gene-targeting frequency in murine embryonic stem (ES) cells with both functional and dysfunctional p53 genes.

The Rad51 gene is the mammalian homologue of the bacterial RecA gene and catalyses homologous recombination in mammalian cells. In some cell types Rad51 has been shown to interact with p53, leading to inhibition of Rad51 activity. Here, we show a two- to four-fold increase in gene-targeting frequency at the HPRT locus using murine ES clones preengineered to overexpress Rad51, and a twofold increase in targeting frequency when a Rad51 expression cassette was cointroduced to wild-type ES cells with the targeting construct. In addition to its effect on homologous recombination, we show that Rad51 may down-regulate illegitimate recombination. We investigated the dependence of these phenomena upon p53 and found no evidence that the Rad 51-mediated increase is affected by the functional status of p53, a conclusion supported by the observed cytoplasmic localisation of p53 in ES cells following electroporation. Furthermore, in the absence of additional Rad51, p53-deficient ES cells do not have elevated rates of homologous recombination with extrachromosomal DNA. These findings demonstrate that Rad51 levels modify both homologous and illegitimate recombination, but that these phenomena are independent of p53 status.

Animals↗
PubMed2003

Effects of donor oocytes and culture conditions on development of cloned mice embryos.

Mice have been successfully cloned from somatic and embryonic stem (ES) cells using the "Honolulu method." In the present study, different donor oocytes and different culture conditions were compared to evaluate the developmental potential of nuclear transfer embryos reconstructed with an inbred ES cell line HM-1. Oocytes were recovered from two different F1 donors B6D2F1 (C57BL/6 x DBA/2) and B6CBAF1 (C57BL/6 x CBA). There was no effect of oocyte origin on development of cloned embryos to the morulae/blastocyst stage (B6D2F1 44.1% vs. B6CBAF1 45.0%), and the transferred embryos could develop to term. Two culture conditions were compared to show their ability to support development to the morulae/blastocyst stage of reconstructed embryos with B6D2F1 oocytes. The total cell number in the cloned blastocysts cultured in M16 with 20% oxygen was much higher than that observed in CZB with 20% oxygen. Low oxygen concentration during culture of nuclear transfer embryos in CZB medium showed no beneficial effect on pre-implantation development, no embryos developed to term after transfer to surrogate mothers. Our results demonstrated that not only B6D2F1, but B6CBAF1 oocytes, can be used for nuclear transfer. M16 medium is superior for culture of nuclear transfer embryos and low oxygen concentration with CZB medium during culture shows no benefit on development of cloned embryos.

Animals↗
PubMed2003

Isolation of murine embryonic stem and embryonic germ cells by selective ablation.

The isolation of murine embryonic stem (ES) cells has been almost exclusively from the 129 mouse strain. Other mouse strains, such as CBA, have proven refractory to ES cell isolation by conventional means. We previously reported the isolation of 87.5% CBA ES lines by selective ablation of differentiating cells (McWhir et al., 1996). Here, we report the isolation of ES and EG cells from 94% CBA embryos hemizygous and homozygous for a neomycin-resistance transgene under the transcriptional control of the Oct3/4 promoter (Oct/neo). Since expression of the Oct/neo transgene only confers drug resistance to undifferentiated cells of the inner cell mass, selection results in the ablation of differentiating cells from the culture. The efficiency of ES isolation by selective ablation in homozygotes is twice that in heterozygotes. ES isolation frequency in permissive strain 129 embryos is enhanced by treatment with an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway but this effect is not sufficient to permit ES isolation from the CBA strain.

Animals↗
PubMed2003

Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Adenoviridae↗
PubMed2003

In vitro osteogenic differentiation of human ES cells.

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.

Adult↗
PubMed2003

Effect of cell confluence on production of cloned mice using an inbred embryonic stem cell line.

Mice have been successfully cloned from both somatic cells and hybrid embryonic stem (ES) cells. Heterozygosity of the donor ES cell genome has been suggested as a crucial factor for long-term survival of cloned mice. In the present study, an inbred ES cell line, HM-1 (129/Ola), and a well-tested ES cell line, R1 (129/Sv x 129/Sv-CP), were used as donor cells to evaluate the developmental potential of nuclear transfer embryos. We found that ES cell confluence dramatically affects the developmental potential of reconstructed embryos. With the ES cell line HM-1 and 80-90% confluence, 49% of reconstructed embryos developed to the morula/blastocyst stage, 9% of these embryos developed to live pups when transferred to the surrogate mothers, and 5 of 18 live pups survived to adulthood. By contrast, at 60-70% confluence, only 22% of embryos developed to the morula/blastocyst stage, and after transfer, only a single fetus reached term. Consistent with previous reports, the nuclei of R1 ES cells were also shown to direct development to term, but no live pups were derived from cells at later passages (>20). Our results show that the developmental potential of reconstructed embryos is determined by both cell confluence and cell passage. These results also demonstrate that the inbred ES cell line, HM-1, can be used to produce viable cloned mice, although less efficiently than most heterozygous ES cell lines.

Animals↗
PubMed2002

Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro.

Primary mouse brain cells were cultured with HPRT (hypoxanthine phosphoribosyl transferase)-deficient ES (embryonic stem) cells to see if the ES cells could provide cues sufficient to reprogram a pluripotential state. After 5 days of coculture, HPRT-deficient ES cells were killed by selection in HAT (hypoxanthine, aminopterin, thymidine) medium. We observed islands of HAT-resistant ES-like cells surrounded by differentiated cells. Cell lines generated from three such "islands" proved to be spontaneous, pluripotential ES-neural hybrids, and gave rise to a chimera following blastocyst injection. Re-expression of the ES-specific gene Foxd3 from somatic-derived chromosomes suggested that the somatic nucleus had been reprogrammed. Our results raise the intriguing possibility that ASCs shown to contribute to multiple tissues in blastocyst-injection studies may not contribute as a result of pluripotency. Instead contributions may arise from spontaneous fusion events in which phenotype is determined by either cytoplasmic dominance, nuclear reprogramming, or both.

Animals↗