Effect of Gsk3 inhibitor CHIR99021 on aneuploidy levels in rat embryonic stem cells

Germline competent embryonic stem (ES) cells can serve as a tool to create genetically engineered rat strains used to elucidate gene function or provide disease models. In optimum culture conditions, ES cells are able to retain their pluripotent state. The type of components present and their concentration in ES cell culture media greatly influences characteristics of ES cells including the ability to maintain the cells in a pluripotent state. We routinely use 2i media containing inhibitors CHIR99021 and PD0325901 to culture rat ES cells. CHIR99021 specifically inhibits the Gsk3β pathway. We have found that the vendor source of CHIR99021 has a measurable influence on the level of aneuploidy seen over time as rat ES cells are passaged. Karyotyping of three different rat ES cell lines passaged multiple times showed increased aneuploidy when CHIR99021 from source B was used. Mass spectrometry analysis of this inhibitor showed the presence of unexpected synthetic small molecules, which might directly or indirectly cause increases in chromosome instability. Identifying these molecules could further understanding of their influence on chromosome stability and indicate how to improve synthesis of this media component to prevent deleterious effects in culture

Mouse chromosome 10

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47005/1/335_2004_Article_BF00360836.pd

Evidence that two phenotypically distinct mouse PKD mutations, bpk and jcpk, are allelic

Evidence that two phenotypically distinct mouse PKD mutations, bpk and jcpk, are allelic. Numerous mouse models of polycystic kidney disease (PKD) have been described. All of these diseases are transmitted as single recessive traits and in most, the phenotypic severity is influenced by the genetic background. However, based on their genetic map positions, none of these loci appears to be allelic and none are candidate modifier loci for any other mouse PKD mutation. Previously, we have described the mouse bpk mutation, a model that closely resembles human autosomal recessive polycystic kidney disease. We now report that the bpk mutation maps to a 1.6 CM interval on mouse Chromosome 10, and that the renal cystic disease severity in our intersubspecific intercross progeny is influenced by the genetic background. Interestingly, bpk co-localizes with jcpk, a phenotyp-ically-distinct PKD mutation, and complementation testing indicates that the bpk and jcpk mutations are allelic. These data imply that distinct PKD phenotypes can result from different mutations within a single gene. In addition, based on its map position, the bpk locus is a candidate genetic modifier for jck, a third phenotypically-distinct PKD mutation

Coat color chimeras and their offspring.

<p>(A) Chimeric animals (albino patches on face) from F344-Tg.EC4011 ES cell injections into SD X DA blastocysts. (B) Offspring from chimeric animal breeding.</p

Breeding results of chimeric animals derived from rat ES cell line F344-Tg.EC4011.

<p>Breeding results of chimeric animals derived from rat ES cell line F344-Tg.EC4011.</p

ES cell morphology and karyotype.

<p>The morphology and karyotype of F344-Tg.EC4011 is shown and is representative of the other ES cell lines. (A) Phase contrast image shows cultured ES cells forming compact colonies with smooth edges. (B) Fluorescence microscopy image of same field of view as (A). Cultured ES cells express the EGFP transgene. Scale bar represents 100 µm. (C) RT-PCR analysis of <i>Oct4</i>, <i>Nanog</i>, and <i>Sox2</i> gene expression using rat specific primers. DAc8, a proven germline competent rat ES cell line (Li et al., 2008) is included as a positive control; rat embryonic fibroblasts (REFs), mouse embryonic fibroblasts (MEFs) as well as a no template control (NTC) are also shown. (D) Cytogenetic analysis. ES cells have a normal male karyotype (42, XY).</p

16S rRNA amplicon sequencing dataset for conventionalized and conventionally raised zebrafish larvae

Data presented here contains metagenomic analysis regarding the sequential conventionalization of germ-free zebrafish embryos. Zebrafish embryos that underwent a germ-free sterilization process immediately after fertilization were promptly exposed to and raised to larval stage in conventional fish water. At 6 days postfertilization (dpf), these “conventionalized” larvae were compared to zebrafish larvae that were raised in conventional fish water never undergoing the initial sterilization process. Bacterial 16S rRNA amplicon sequencing was performed on DNA isolated from homogenates of the larvae revealing distinct microbiota variations between the two groups. The dataset described here is also related to the research article entitled “Microbial modulation of behavior and stress responses in zebrafish larvae” (Davis et al., 2016) [1]. Keywords: Microbiome, Microbiota, Zebrafish larvae, 16S rRNA sequencing, Gnotobioti