Jason G. Knott

Cloned transchromosomic calves producing human immunoglobulin

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (λ) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78–100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

SWI/SNF-Brg1 Regulates Self-Renewal and Occupies Core Pluripotency-Related Genes in Embryonic Stem Cells

The SWI/SNF‐Brg1 chromatin remodeling protein plays critical roles in cell‐cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self‐renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self‐renewal and pluripotency. RNA interference‐mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self‐renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome‐wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency‐related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co‐occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self‐renewal and pluripotency in ES cells. STEM CELLS 2009;27:317–328

Production of calves from G1 fibroblasts.

Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a “shake-off” procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the “shake-off” treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from “shake-off” cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.

Effect of Fibroblast Donor Cell Age and Cell Cycle on Development of Bovine Nuclear Transfer Embryos In Vitro

Abstract The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6–15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2–3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.

Generation of Leukemia Inhibitory Factor and Basic Fibroblast Growth Factor-Dependent Induced Pluripotent Stem Cells from Canine Adult Somatic Cells

For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.

Brg1 Is Required for Cdx2-Mediated Repression of Oct4 Expression in Mouse Blastocysts

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.

Fertilization and Inositol 1,4,5-Trisphosphate (IP3)-Induced Calcium Release in Type-1 Inositol 1,4,5-Trisphosphate Receptor Down-Regulated Bovine Eggs

Abstract It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP3) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca2+]i) seen during mammalian fertilization. IP3 promotes Ca2+ release in eggs by binding to its receptor, the type-1 IP3 receptor (IP3R-1, also known as ITPR1), a ligand-gated Ca2+ channel located in the membrane of the endoplasmic reticulum, the main Ca2+ store of the cell. While IP3R-1 has been shown to mediate all Ca2+ release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca2+ release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a ∼70%–80% reduction in the number of intact IP3R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP3 analogue, adenophostin A. Functional Ca2+ release analysis revealed that IP3R-1 is the predominant Ca2+ release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca2+]i oscillations in response to fertilization, expression of PLCζ (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP3 and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca2+]i oscillations in this reduced IP3R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP3R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP3R-1-mediated Ca2+ release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca2+]i oscillations at the level of IP3R-1.

Release of the Ca2+ oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.

Transcription factor AP-2γ induces early Cdx2 expression and represses HIPPO signaling to specify the trophectoderm lineage

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling. Summary: Mouse trophectoderm specification is regulated by AP-2γ/TFAP2C, which activates Cdx2 expression, controls actin dynamics and suppresses HIPPO signaling via multiple pathways, including the potentiation of cell polarity.

Transcription factor AP-2γ is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis

The trophectoderm epithelium is the first differentiated cell layer to arise during mammalian development. Blastocyst formation requires the proper expression and localization of tight junction, polarity, ion gradient and H2O channel proteins in the outer cell membranes. However, the underlying transcriptional mechanisms that control their expression are largely unknown. Here, we report that transcription factor AP-2γ (Tcfap2c) is a core regulator of blastocyst formation in mice. Bioinformatics, chromatin immunoprecipitation and transcriptional analysis revealed that Tcfap2c binds and regulates a diverse group of genes expressed during blastocyst formation. RNA interference experiments demonstrated that Tcfap2c regulates genes important for tight junctions, cell polarity and fluid accumulation. Functional and ultrastructural studies revealed that Tcfap2c is necessary for tight junction assembly and paracellular sealing in trophectoderm epithelium. Aggregation of control eight-cell embryos with Tcfap2c knockdown embryos rescued blastocyst formation via direct contribution to the trophectoderm epithelium. Finally, we found that Tcfap2c promotes cellular proliferation via direct repression of p21 transcription during the morula-to-blastocyst transition. We propose a model in which Tcfap2c acts in a hierarchy to facilitate blastocyst formation through transcriptional regulation of core genes involved in tight junction assembly, fluid accumulation and cellular proliferation.