Methods in Molecular Biology

IQCELL is a platform that infers Boolean gene regulatory networks from single-cell RNA sequencing data. Boolean networks can be simulated under normal and perturbed conditions. In this chapter, we provide a detailed guideline for implementing IQCELL from a raw dataset. The steps include processing data, inferring informative genes, inferring gene regulatory network, and simulating the resulted network under normal and perturbed conditions.

Diabetes mellitus can be categorized as one of the prolonged metabolic disorders that are associated with inappropriately elevated blood glucose levels. Among the subgroups of this disease, type 2 diabetes accounts for the most patients. Although pharmaceutical and non-pharmaceutical treatments have been employed to control the progression of the disease, as with any treatment approach, both therapeutic approaches are associated with side effects and challenges. Nowadays, the emergence of treatment methods based on stem cells has attracted the attention of researchers in order to treat diabetes fundamentally and provide a long-term solution...

Human pluripotent stem cells (hPSCs) form an ideal system to study the formation of placental cells, from an undifferentiated human embryonic stem cell state. The conventional human in vitro model systems to study the human placenta cannot be employed for understanding placental dysfunctions or the development of specialized placental cell types. Hence, human PSCs make an ideal model system to study human placental development and disorders. Here, we describe an efficient and validated protocol to reproducibly study the formation of human cytotrophoblasts (CTBs) and syncytiotrophoblast (STBs) from undifferentiated hPSCs...

Amyotrophic lateral sclerosis (ALS) is a progressive and degenerative disorder of the nervous system that can significantly reduce the physical activity of patients at the end stages. As the field of disease pathophysiology has advanced in recent years, studies have looked at the role of neuromuscular junction's dysfunctionality in ALS. In the past years, various in vitro and in vivo models were developed to scrutinize the underlying mechanisms of the disease and investigate the effects of candidate drugs, but the application of the developed models faced many challenges...

Tracing the fate of individual cells and their progeny is necessary and significant for stem cell research and cancer research. Changes in lineage-specific transcription factor levels during lineage commitment are gradual and continuous. Development of single-cell sequencing technology allows many different states of cells to be sequenced at an unprecedented resolution, and it has been proved that single-cell transcriptomics meets lineage tracing. Here, we introduce a detailed protocol for the lineage tracing by single-cell transcriptomics to clarify the dynamics of lineage commitment...

Hematopoiesis is maintained throughout life from the hematopoietic stem cell niche in which hematopoietic stem cells and lineage-specific hematopoietic progenitors (HSPCs) reside and regulate hematopoiesis. Meanwhile, HSPCs behavior is modulated by both cell intrinsic (e.g., transcriptional factors) and cell extrinsic (e.g., cytokines) factors. Dysregulation of these factors can alter HSPCs function, leading to disrupted hematopoiesis, cellular changes, and subsequent hematological diseases and malignancies...

The microfluidic amniotic sac embryoid (μPASE) is a human pluripotent stem cell (hPSC)-derived multicellular human embryo-like structure with molecular and morphological features resembling the progressive development of the early post-implantation human embryonic sac. The microfluidic device is specifically designed to control the formation of hPSC clusters and expose the clusters to different morphogen environments, allowing the development of μPASEs in a highly controllable, reproducible, and scalable fashion...

Stem cell-derived embryos in vitro allow the exploration of the very early stages of human embryogenesis in vitro and are thus promising for widespread applications in developmental biology, related developmental disease modeling, and drug discovery. Several cell resources have been utilized, with different efficiencies and methods for generating human blastoids, a structure similar to natural blastocysts. Human EPS cells were reported to contribute to the embryonic and extraembryonic lineages and therefore can be a practical and efficient cell resource for constructing human blastoids...

Parkinson's disease is a progressive neurodegenerative disorder, which is mainly characterized by unintended or uncontrollable body movements. Pathophysiologically, disturbances in the neurotransmission system of the brain like dopaminergic system and synaptic dysfunction are classified as top-rated causes of the onset of Parkinson's disease, which symptoms can be different according to the involvement of neurotransmission system type and the effect of the disease on the motor and non-motor systems. Although some pharmacological and non-pharmacological approaches have been applied to control and slow down the progression of the disease, a definitive cure has not yet been discovered...

Salivary gland myoepithelial cells regulate salivary secretion and have been implicated in the histological diversity of salivary gland tumors. However, isolation of myoepithelial cells has been difficult owing to a lack of detailed functional analysis and cell surface markers. Therefore, we aimed to isolate myoepithelial cells from adult mouse submandibular glands using the epithelial marker EpCAM and cell adhesion factor CD49f as indicators and characterize them via sphere-forming culture. Functional analysis of specific gene expression in myoepithelial cells is possible via cell transfection experiments using the piggyBac transposon vector system...

Genome editing technology facilitates the creation of specific and precise genetic changes to unravel gene function and rapidly transfer unique alleles between chicken breeds in contrast to lengthy traditional crossbreeding methods for the study of poultry genetics. Innovations in genome sequencing technology have made it possible to map polymorphisms associated with both monogenic and multigenic traits in livestock species. We, and many others, have demonstrated the use of genome editing to introduce specific monogenic traits in chicken through targeting of cultured primordial germ cells...

The generation of genetically engineered (GE) pigs for disease modeling and xenotransplantation has been massively facilitated by the discovery of the CRISPR/Cas9 system. For livestock, genome editing is a powerful tool when used in combination with either somatic cell nuclear transfer (SCNT) or microinjection (MI) into fertilized oocytes. To generate either knockout or knock-in animals using SCNT, genome editing is carried out in vitro. This has the advantage that fully characterized cells are being employed to generate cloned pigs, predetermining their genetic makeups...

Pluripotent stem cell (PSC) injection to the blastocyst stage embryos is a widely used method to evaluate the pluripotency through chimeric contribution. It is routinely used to produce transgenic mice. However, PSC injection to the blastocyst stage embryos in rabbits is challenging. At this stage, the in vivo developed rabbit blastocysts possess a thick mucin layer that is inhibitory for microinjection, whereas in vitro developed rabbit blastocysts that lack such mucin layer often fail to implant after embryo transfer...

The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.

The availability of reliable germline competent rat embryonic stem cell (ESC) lines that can be genetically manipulated provides an important tool for generating new rat models. Here we describe the process for culturing rat ESCs, microinjecting the ESCs into rat blastocysts, and transferring the embryos to surrogate dams by either surgical or non-surgical embryo transfer techniques to produce chimeric animals with the potential to pass on the genetic modification to their offspring.

Rat germline-competent embryonic stem (ES) cell lines have been available since 2008, and rat models with targeted mutations have been successfully generated using ES cell-based genome targeting technology. This chapter will focus on the procedures of gene targeting in rat ES cells.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI)...

The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes...

CRISPR/Cas9 technology is a versatile tool for engineering biology that has dramatically transformed our ability to manipulate genomes. In this protocol, we use its capacity to generate two double-strand breaks simultaneously, at precise positions in the genome, to generate mouse or rat lines with deletion, inversion, and duplication of a specific genomic segment. The technic is called CRISMERE for CRISpr-MEdiated REarrangement. This protocol describes the different steps to generate and validate the different chromosomal rearrangements that can be obtained with the technology...

Genetic engineering in the rat has been revolutionized by the development of CRISPR-based genome editing tools. Conventional methods for inserting genome editing elements such as CRISPR/Cas9 reagents into rat zygotes include cytoplasmic or pronuclear microinjections. These techniques are labor-intensive, require specialized micromanipulator equipment, and are technically challenging. Here, we describe a simple and effective method for zygote electroporation in which CRISPR/Cas9 reagents are introduced into rat zygotes via pores produced by precise electrical pulses applied to the cells...