Osteoclast Differentiation Assay.
Abstract Osteoclasts are highly specialized multinucleated cells derived from the monocyte/macrophage hematopoietic lineage that are uniquely capable of adhering to bone matrix and resorbing bone. The tartrate-resistant acid phosphatase (TRAP) assay is the most common method to detect osteoclasts population in vitro. Here we described a general protocol of inducing osteoclast differentiation from the murine macrophage cell line, RAW264.7, and identification of osteoclasts with the classical TRAP assay. PMID: 30378050 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Yang J, Bi X, Li M Tags: Methods Mol Biol Source Type: research

Primary Cultures for Pancreatic Stellate Cells (PSCs).
Abstract Primary cultures of pancreatic stellate cells (PSCs) remain an important basis for in vitro study. However, effective methods for isolating abundant PSCs are currently lacking. This purpose of this chapter is to report our novel approach to isolating PSCs from normal rat pancreas and human pancreatic ductal adenocarcinoma (PDAC) tissue. Normal PSCs were isolated with enzyme digestion and ladder centrifugation with Nycodenz solution. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrow method from fresh human PDAC tissues. Isolated ac...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Tian L, Lu Z, Miao Y Tags: Methods Mol Biol Source Type: research

Cytokine Profiling and Orthotopic Xenografing of Pancreatic Stellate Cells.
Abstract Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the most lethal human malignancies with a poor prognosis due to systemic metastasis and a high recurrence rate. Interactions between tumor and stromal cells play a critical role in tumor progression. However, the interaction between PSCs and pancreatic cancer cells (PCCs) and the underlying mechanisms are poorly understood. Coculture system with PSCs and PCCs is very useful technique platform for the in vitro and in vivo study of the interaction between these two cellular components. In this protocol, we aim to describe the cytokine profiling ...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Qian D, Tian L, Lu Z, Miao Y Tags: Methods Mol Biol Source Type: research

Quantitative Method to Track Proteolytic Invasion in 3D Collagen.
Abstract Since many tumors are associated with a pronounced collagen-rich stromal reaction, there is increasing interest in understanding mechanisms by which cancer cells invade through the collagen barrier. Here we describe a quantitative method to track cell invasion in 3D collagen I gels. We analyze invasion by quantifying proteolytic tracks generated by invading cancer cells through a 3D collagen microenvironment. We provide a detailed protocol for this quantitative assay, which can be used to characterize signaling pathways that regulate invasion in the 3D microenvironment. PMID: 30378053 [PubMed - in pr...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Ebine K, Chow CR, Munshi HG Tags: Methods Mol Biol Source Type: research

Detection and Quantification of Macropinosomes in Pancreatic Tumors.
Abstract Macropinocytosis is a mechanism of fluid-phase endocytosis that functions in the nonspecific internalization of extracellular fluid. This uptake pathway has specialized roles in different cell types and organisms, and its importance has recently been established in several diseases, including cancer. In cancer, macropinocytosis is stimulated by oncogenes, such as Ras, and macropinocytic cargo is targeted to lysosomes for degradation, providing a catabolic route for tumor cells to obtain amino acids from the tumor microenvironment. Here, we describe a protocol to employ fluorescently labeled...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Lee SW, Alas B, Commisso C Tags: Methods Mol Biol Source Type: research

Methods for Monitoring Macroautophagy in Pancreatic Cancer Cells.
Abstract Macroautophagy is a catabolic process through which redundant, aged, or damaged cellular structures are first enclosed within double-membrane vesicles (called autophagosomes), and thereafter degraded within lysosomes. Macroautophagy provides a primary route for the turnover of macromolecules, membranes and organelles, and as such plays a major role in cell homeostasis. As part of the stress response, autophagy is crucial to determine the cell fate in response to extracellular or intracellular injuries. Autophagy is involved in cancerogenesis and in cancer progression. Here we illustrate the essential meth...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Vidoni C, Ferraresi A, Seca C, Secomandi E, Isidoro C Tags: Methods Mol Biol Source Type: research

Measurement of Reactive Oxygen Species by Fluorescent Probes in Pancreatic Cancer Cells.
Abstract Pancreatic cancer is a highly lethal disease and is projected to become the second leading cause of cancer-related death by 2020. Among the different subtypes, pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. The genetic landscape of PDAC shows nearly ubiquitous mutations of KRAS. However, expression of KRAS somatic mutants alone is insufficient to drive PDAC. Redox deregulation may contribute significantly to KRAS-mediated PDAC. Thus, measurement of cellular reactive oxygen species (ROS) levels is essential to determine how oxidative stress affects mutant KRAS and mod...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Luo Y, Wang D, Abbruzzese JL, Lu W Tags: Methods Mol Biol Source Type: research

Evaluating the Metabolic Alterations in Pancreatic Cancer.
Abstract Metabolic reprograming is an established hallmark of cancer cells. Pancreatic cancer cells, by virtue of the underlying oncogenic drivers, demonstrate metabolic reprograming to sustain growth, invasiveness, and therapy resistance. The increased demands of the growing tumor cells alter the metabolic and signaling pathways to meet the growing nutrient requirements. Investigating the metabolic vulnerabilities of tumor cells can help in developing effective therapeutics to target pancreatic cancer. In this chapter, we explain in detail the methods to evaluate the metabolic changes occurring in the tumor. This...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Dasgupta A, Shukla SK, Gunda V, King RJ, Singh PK Tags: Methods Mol Biol Source Type: research

Isolation of Extracellular Vesicles for Cancer Diagnosis and Functional Studies.
Abstract Extracellular vesicles (EVs) are a diverse category of cellular export products that are present in a variety of biofluids and cell culture media. EVs contain a wide variety of macromolecules that represent a sampling of the cytoplasmic or endosomal compartments and function in cell-to-cell paracrine and endocrine signaling; it has been demonstrated that pathological states such as oxidative stress, transformation, apoptosis, and various cell injuries induce cells to increase their EV release rate, simultaneously altering their composition to reflect the altered state of the cellular origin. Specifically,...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Brenner AW, Su GH, Momen-Heravi F Tags: Methods Mol Biol Source Type: research

Laser Capture Microdissection on Frozen Sections for Extraction of High-Quality Nucleic Acids.
We present here an optimized method to retrieve intact RNA from laser capture microdissected tissue samples, using pancreatic ductal adenocarcinoma as an example, in order to separately profile tumor epithelial and stromal compartments. This method may also be applied to nonmalignant tissues to isolate cellular samples from any morphologically identifiable structure. PMID: 30378061 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Maurer HC, Olive KP Tags: Methods Mol Biol Source Type: research

Induction of Pancreatic Inflammation Accelerates Pancreatic Tumorigenesis in Mice.
Abstract Pancreatitis is a major risk factor for the development of pancreatic cancer. In genetically engineered mouse models, induction of pancreatic inflammation dramatically accelerates oncogenic KRas-induced fibrosis, precancerous PanIN formation, and tumorigenesis. Here we describe simple methods of secretagogue-induced experimental acute and chronic pancreatitis, the most commonly used pancreatitis models, and their applications in pancreatic cancer research. Additionally, the preparation of primary pancreatic acinar cells is introduced. Primary acinar cells can be used to study the early events of pancreati...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Zhuang L, Zhan X, Bi Y, Ji B Tags: Methods Mol Biol Source Type: research

Pancreatic Acinar-to-Ductal Metaplasia and Pancreatic Cancer.
Abstract Acinar-to-ductal metaplasia (ADM) of the pancreas is a process that pancreatic acinar cells differentiate into ductal-like cells with ductal cell traits. The metaplasia of pancreatic acinar cells manifests their ability to adapt to the genetic and environmental pressure they encounter. However, with oncogenic genetic insults and/or sustained environmental stress, ADM may lead to pancreatic intraepithelial neoplasia (PanIN), which is a common precancerous lesion that precedes pancreatic cancer. Understanding the intermediate states of ADM and important molecules that regulate ADM formation may help the dev...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Wang L, Xie D, Wei D Tags: Methods Mol Biol Source Type: research

Molecular and Physiological Evaluation of Pancreatic Cancer-Induced Cachexia.
Abstract Cachexia, a complex metabolic syndrome, is characterized by involuntary weight loss along with muscle wasting and fat depletion leading to poor quality of life of patients. About 80% of pancreatic cancer patients exhibit cachectic phenotype at the time of diagnosis. Here, we present the several molecular and physiological parameters, which we utilize to study the pancreatic cancer-induced cachexia in in vitro models and preclinical mice models of pancreatic cancer. We have described myotube and adipocyte-based in vitro models of muscle and fat wasting, including methods of cell culture, differentiati...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Shukla SK, Dasgupta A, Mulder SE, Singh PK Tags: Methods Mol Biol Source Type: research

Computational Methods for Identification of T Cell Neoepitopes in Tumors.
Abstract Cancer immunotherapy has experienced several major breakthroughs in the past decade. Most recently, technical advances in next-generation sequencing methods have enabled discovery of tumor-specific mutations leading to protective T cell neoepitopes. Many of the successes are enabled by computational methods, which facilitate processing of raw data, mapping of mutations, and prediction of neoepitopes. In this book chapter, we provide an overview of the computational tasks related to the identification of neoepitopes, propose specific tools and best practices, and discuss strengths, weaknesses, and future c...
Source: Mol Biol Cell - November 2, 2018 Category: Molecular Biology Authors: Jurtz VI, Olsen LR Tags: Methods Mol Biol Source Type: research

Purification of Myogenic Progenitors from Human Muscle Using Fluorescence-Activated Cell Sorting (FACS).
Abstract Primary myoblasts derived from human tissue are a valuable tool in research of muscle disease and pathophysiology. However, skeletal muscle biopsies, especially from diseased muscle, contain a plethora of non-myogenic cells, necessitating purification of the myogenic cell population. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study. PMID: 30367405 [PubMed ...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Pakula A, Spinazzola JM, Gussoni E Tags: Methods Mol Biol Source Type: research

Electrical Pulse Stimulation of Primary Human Skeletal Muscle Cells.
Abstract Electrical pulse stimulation (EPS) is an in vitro method of inducing contractions in cultured skeletal muscle cells of human and animal origin. Motor neuron activation of muscle fibers can be replaced by applying EPS on differentiated skeletal muscle cells (myotubes) in culture (Thelen et al. Biochemical J 321:845-848, 1997, Fujita et al. Exp Cell Res 313:1853-1865, 2007).Here we describe two protocols for EPS of human myotubes in 6-well plates: acute, high-frequency (single bipolar pulses of 2 ms, 100 Hz for 200 ms every fifth second for 5-60 min, 10-30 V) and chronic, low-freque...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Nikolić N, Aas V Tags: Methods Mol Biol Source Type: research

Transdifferentiation of Muscle Satellite Cells to Adipose Cells Using CRISPR/Cas9-Mediated Targeting of MyoD.
Abstract Brown adipocytes dissipate energy through non-shivering thermogenesis mediated by UCP1 protein, hence representing a powerful target to overcome obesity due to energy surplus. However, brown adipocytes are scarce in adult humans, especially in obese subjects, urging the development of novel strategies to boost the number of these thermogenic adipocytes from a therapeutical perspective. In this regard, transdifferentiation of myoblasts into brown adipocytes represents a promising approach. Here, we describe a method that we have recently developed to transdifferentiate myoblasts into brown adipocytes throu...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Chen J, Wang C, Kuang S Tags: Methods Mol Biol Source Type: research

Chromatin Immunoprecipitation in Skeletal Myoblasts.
Abstract Chromatin immunoprecipitation (ChIP) is a powerful and sensitive technique that is widely used to study DNA-protein interactions. It enables an unbiased genome-wide analysis of transcriptional changes during several biological processes including cellular differentiation. Here, we describe a step-by-step protocol to identify histone modifications, transcription factor, and co-factor binding to chromatin in skeletal myoblasts. We discuss critical steps during cell harvesting, sonication, and immunoprecipitation and provide notes to evade common pitfalls. PMID: 30367408 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Rao VK, Shankar SR, Taneja R Tags: Methods Mol Biol Source Type: research

Exercising Bioengineered Skeletal Muscle In Vitro: Biopsy to Bioreactor.
Abstract The bioengineering of skeletal muscle tissue in-vitro has enabled researchers to more closely mimic the in-vivo skeletal muscle niche. The three-dimensional (3-D) structure of the tissue engineered systems employed to date enable the generation of highly aligned and differentiated myofibers within a representative biological matrix. The use of electrical stimulation to model concentric contraction, via innervation of the myofibers, and the use of mechanical loading to model passive lengthening or stretch has begun to provide a manipulable environment to investigate the cellular and molecular responses fol...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Turner DC, Kasper AM, Seaborne RA, Brown AD, Close GL, Murphy M, Stewart CE, Martin NRW, Sharples AP Tags: Methods Mol Biol Source Type: research

Isolation and Purification of Satellite Cells from Young Rats by Percoll Density Gradient Centrifugation.
Abstract Satellite cells (SCs) are myogenic stem cells that play an important role in skeletal muscle regeneration and hypertrophy. Primary cultures of SCs are useful to analyze cell functions; however, it is difficult to obtain highly pure SCs from young rats with the conventional procedures. The purpose of this study is to establish a purification method for SC isolation from young rats and quantitatively evaluate the purification procedure employing Percoll, a common research tool to purify cells. We elucidated the purity of SCs collected by Percoll density gradient centrifugation using real-time RT-qPCR and im...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Matsuyoshi Y, Akahoshi M, Nakamura M, Tatsumi R, Mizunoya W Tags: Methods Mol Biol Source Type: research

Lentivirus-Mediated RNAi in Skeletal Myogenesis.
hen J Abstract RNA interference (RNAi) has greatly facilitated investigation of gene functions in vitro as well as in vivo. Recombinant lentivirus is widely used to deliver small hairpin RNA (shRNA) because of its high transduction capacity into diverse cell types and tissues. Here, we describe methods of lentivirus-mediated delivery of shRNA for the study of skeletal muscle cell differentiation in vitro and injury-induced muscle regeneration in mice. PMID: 30367411 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Kim D, Reyes-Ordoñez A, Chen J Tags: Methods Mol Biol Source Type: research

Coculture Method to Obtain Endothelial Networks Within Human Tissue-Engineered Skeletal Muscle.
Abstract Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenito...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Gholobova D, Gerard M, Terrie L, Desender L, Shansky J, Vandenburgh H, Thorrez L Tags: Methods Mol Biol Source Type: research

Fluorescence-Activated Cell Sorting of Larval Zebrafish Muscle Stem/Progenitor Cells Following Skeletal Muscle Injury.
Abstract This chapter describes a protocol for the isolation of larval zebrafish muscle stem/progenitor cells by fluorescence-activated cell sorting (FACS). This method has been successfully applied to isolate pax3a expressing cells 3 days following needle stab skeletal muscle injury. The cell sorting strategy described here can easily be adapted to any cell type at embryonic or larval stages. RNA extracted from the sorted cells can be used for subsequent downstream applications such as quantitative PCR (qPCR), microarrays, or next generation sequencing. PMID: 30367418 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Ratnayake D, Currie PD Tags: Methods Mol Biol Source Type: research

Myoblast Phosphoproteomics as a Tool to Investigate Global Signaling Events During Myogenesis.
Abstract Protein phosphorylation is a universal covalent chemical modification of amino acids involved in a large number of biological processes including cell signaling, metabolism, proliferation, differentiation, survival/death, ageing, and many more. Regulation of protein phosphorylation is essential in myogenesis and indeed, when the enzymatic activity of protein kinases is distrupted in myoblasts, myogenesis is affected. In this chapter we describe a method to profile the phosphoproteome of myoblasts using mass spectrometry. Phosphate groups are labile and easily lost during the processing of samples for mass...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Jones FK, Hardman GE, Ferries S, Eyers CE, Pisconti A Tags: Methods Mol Biol Source Type: research

Preparation and Culturing of Atlantic Salmon Muscle Cells for In Vitro Studies.
Abstract This chapter outlines methods for isolating myosatellites from Atlantic salmon (Salmo salar), how to keep them in culture and differentiate them into mature myocytes. The protocol further describes how to trans-differentiate the myocytes into osteoblasts (bone cells). PMID: 30367423 [PubMed - in process] (Source: Mol Biol Cell)
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Oestbye TK, Ytteborg E Tags: Methods Mol Biol Source Type: research

RNA Interference Screening for Genes Regulating Drosophila Muscle Morphogenesis.
rer F Abstract RNA interference (RNAi) is the method of choice to systematically test for gene function in an intact organism. The model organism Drosophila has the advantage that RNAi is cell autonomous, meaning it does not spread from one cell to the next. Hence, RNAi can be performed in a tissue-specific manner by expressing short or long inverted repeat constructs (hairpins) designed to target mRNAs from one specific target gene. This achieves tissue-specific knock-down of a target gene of choice. Here, we detail the methodology to test gene function in Drosophila muscle tissue by expressing hairpins in a musc...
Source: Mol Biol Cell - October 28, 2018 Category: Molecular Biology Authors: Kaya-Çopur A, Schnorrer F Tags: Methods Mol Biol Source Type: research

Transgene Recombineering in Bacterial Artificial Chromosomes.
Abstract Bacterial Artificial Chromosome (BAC) libraries are a valuable research resource. Any one of the clones in these libraries can carry hundreds of thousands of base pairs of genetic information. Often the entire coding sequence and significant upstream and downstream regions, including regulatory elements, can be found in a single BAC clone. BACs can be put to many uses, such as to study the function of human genes in knockout mice, to drive reporter gene expression in transgenic animals, and for gene discovery. In order to use BACs for experimental purposes it is often desirable to genetically modify ...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Zeidler MG, Saunders TL Tags: Methods Mol Biol Source Type: research

Generating Genetically Engineered Mice Using a Spermatogonial Stem Cell-Mediated Method.
Abstract Mouse spermatogonial stem cells (SSCs) can be grown in culture for long periods. Cultured SSCs, also called germline stem (GS) cells, maintain themselves by self-renewing proliferation while retaining the ability to differentiate into sperm. Thus, when transplanted into the seminiferous tubules of a host mouse testis, they settle in the basal compartment of the tubules and establish spermatogenenic colonies. The sperm produced in the host are competent to produce offspring. This can be exploited for the generation of genetically modified mice, through the transplantation of genetically modified GS cells. ...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Sato T, Ogawa T Tags: Methods Mol Biol Source Type: research

Chimeric Mouse Generation by ES Cell Blastocyst Microinjection and Uterine Transfer.
Abstract The ability to generate chimeric mice through microinjecting embryonic stem (ES) cells into blastocysts is a critical step for the conventional ES cell-mediated knockout technology. In recent years, designer nuclease-based methods, especially the CRISPR technology, have substantially decreased the needs for blastocyst microinjection. However, this method has still remained as a valuable technique for generating sophisticated genetic models as well as for stem cell research. In this chapter, we describe the detailed procedures used in our laboratory on how to use ES cells to produce chimeric mice, inc...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Du Y, Xie W, Zhang F, Liu C Tags: Methods Mol Biol Source Type: research

Creating Knockin Alleles in Mouse Embryonic Stem Cells by CRISPR/Cas9-Mediated Homologous Recombination Without Drug Selection.
Abstract The rapidly evolving CRISPR/Cas9-mediated genome editing provides the convenience of genome manipulation directly in mouse zygotes for a number of genomic manipulations; but knockins of large insertions prove to be relatively inefficient at least with double-strand DNA as targeting constructs. Here, we describe an alternative approach to the direct genome editing in mouse zygotes by generating knockin alleles in mouse embryonic stem cells first with CRIPSR-mediated homologous recombination. Our results show this approach is efficient and requires no drug selection in mouse embryonic stem cells as in class...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Liu P, Li Y, Lei J, Dong L Tags: Methods Mol Biol Source Type: research

Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.
Abstract NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are an immunodeficient strain that enables human cell xenografts. However, NSG mice possess a complex genetic background that would complicate cross-breeding with other inbred transgenic or knockout mouse strains to establish a congenic strain with a desired genetic modification in the NSG background. Newly developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology enables modification of the mouse genome at the zygote stage without the need for extensive cross-breeding or the use of embryonic stem cells. In this chapter, we use...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Du Y, Xie W, Zhang F, Choi U, Liu C, Sweeney CL Tags: Methods Mol Biol Source Type: research

Generation of CRISPR-Edited Rodents Using a Piezo-Driven Zygote Injection Technique.
Abstract Direct modification of the genome of the zygotes (i.e., one-cell embryos) by the CRISPR/Cas9-editing reagents, followed by embryo transfer to pseudopregnant females for live birth, has been the most effective method to generate laboratory rodent models for research. The method relies on proper delivery of the editing reagents into zygotes, which is commonly achieved by a standard or slightly modified pronuclear microinjection technique. In this chapter, we describe in detail an alternative delivery method, named piezo-driven cytoplasmic microinjection, which offers a superior embryo survival and birth rat...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Scott MA, Hu YC Tags: Methods Mol Biol Source Type: research

Delivery of CRISPR-Cas9 into Mouse Zygotes by Electroporation.
Abstract The CRISPR-Cas9 system in bacteria and archaea has recently been exploited for genome editing in various model organisms, including the mice. In this scheme, components of the CRISPR-Cas9 system are delivered into the mouse zygote and mutant mice carrying genetic modifications derived. Although microinjection has been the technology of choice, electroporation has also emerged and been proven to be effective delivering CRISPR-Cas9 reagents into the mouse zygote. Here, we describe the experimental protocol employing electroporation to deliver CRISPR-Cas9 reagents into mouse embryos and derive gene-edited mu...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Qin W, Wang H Tags: Methods Mol Biol Source Type: research

Generation of Conditional Knockout Mice by Sequential Insertion of Two loxP Sites In Cis Using CRISPR/Cas9 and Single-Stranded DNA Oligonucleotides.
Abstract Conditional knockout (cKO) mice are extremely valuable for biomedical research because they enable detailed analyses of gene functions in a tissue- or temporally-specific fashion. The conventional method for generating cKO mice is time consuming and labor intensive, which involves making a large gene-targeting construct, transfecting and screening many embryonic stem (ES) cell clones, injecting positive ES clones into blastocysts to produce chimeric mice, and breeding the chimeras to transmit the targeted gene through the germline. Recently developed CRISPR technology has revolutionized the way geneticall...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Liu Y, Du Y, Xie W, Zhang F, Forrest D, Liu C Tags: Methods Mol Biol Source Type: research

Improvement of Mouse Cloning from Any Type of Cell by Nuclear Injection.
Abstract Somatic cell nuclear transfer (SCNT) technology has become a useful tool for animal cloning, gene manipulation, and genomic reprograming research. The original SCNT was performed using cell fusion between the donor cell and oocyte. This method remains very popular, but we have recently developed an alternative method that relies on nuclear injection rather than cell fusion. The advantages of nuclear injection include a shortened experimental procedure and reduced contamination of donor cytoplasm in the oocyte. In particular, only this method allows us to perform SCNT using dead cells or naked nuclei such ...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Wakayama S, Kishigami S, Wakayama T Tags: Methods Mol Biol Source Type: research

Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line.
Abstract The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteri...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Sakurai T, Kamiyoshi A, Ohtsuka M, Gurumurthy CB, Sato M, Shindo T Tags: Methods Mol Biol Source Type: research

Molecular Aspects of Zinc Finger Nucleases (ZFNs)-Mediated Gene Editing in Rat Embryos.
Abstract This chapter contains a collection of protocols involved in using ZFNs to create rat models with various types of genome editing, including simple knockout, point mutation, large deletions, floxing, and insertions. The protocols cover ZFN and donor design criteria, in vitro transcription of ZFNs, validation of ZFNs activity in cultured cells, RNA stability test, microinjection sample preparation, genotyping and in vitro confirmation of floxed alleles, and Southern blot analysis, most of which are not limited to using ZFNs. Instead they apply to model creation in general. When appropriate, a comparison bet...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Cui X Tags: Methods Mol Biol Source Type: research

Organ Generation from Knockedout Rat Blastocysts Complemented with Pluripotent Stem Cells.
Abstract Regeneration of human organs in domestic animal model would provide enough number of functional donor organs in transplantation therapy. Recent progresses in pluripotent stem cells and nuclease-based genome editing tools have set the stage for investigating the chimeric complementation approach to generate functional organs from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. In this chapter, protocol for allogeneic or xenogeneic organ generation using knocked-out (KO) rat blastocysts and the rat or mouse ES/iPS cells is described. The protocol includes (1) the preparation of KO rat col...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Hirabayashi M, Hochi S Tags: Methods Mol Biol Source Type: research

Generation of Rabbit Models by Gene Editing Nucleases.
Abstract Due to the lack of germline transmitting pluripotent stem cells (PSCs) cell lines and the extreme difficulty of somatic cell nuclear transfer (SCNT) in rabbit, the gene targeting technology in rabbit was lagging far behind those in rodents and in farm animals. As a result, the development and application of genetically engineered rabbit model are much limited. With the advent of gene editing nucleases, including ZFN, TALEN, and CRISPR/Cas9, it is now possible to produce gene targeting (i.e., knockout, knockin) rabbits with high success rates. In this chapter, we describe a comprehensive, step-by-step prot...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Yang D, Xu J, Chen YE Tags: Methods Mol Biol Source Type: research

Production of Genetically Engineered Porcine Embryos by Handmade Cloning.
Abstract Genetic engineering is essential to realize the full potentials of pigs both as livestock and as animal models of human disease. With the development of new genetic engineering technologies, such as the clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system, the porcine genome can be engineered with high efficiency. In this chapter, we describe a protocol in employing the CRISPR/Cas9 system to genetically engineer the porcine genome in fibroblast cells, the procedures to establish single-cell-derived porcine fibroblast cell colonies carrying the desired ge...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Li R, Miao J, Wang Z Tags: Methods Mol Biol Source Type: research

Electrofusion of 2-Cell Embryos for Porcine Tetraploid Embryo Production.
Abstract The electrofusion of 2-cell embryos proves to be a simple and efficient way of generating mammalian tetraploid embryos. Many factors affect the fusion efficiency, such as fusion medium, electric field intensity, and fusion pulse length. In mice, production of tetraploid embryos by electrofusion has already been investigated; however, the investigation to produce porcine tetraploid embryos is seldom reported. In this chapter, we will describe oocytes in vitro maturation, in vitro fertilization, and the optimum conditions for electrofusion of 2-cell embryos to produce tetraploid embryos in pig. PMID: 3...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Kong Q, Liu Z Tags: Methods Mol Biol Source Type: research

Gene Knockouts in Goats Using CRISPR/Cas9 System and Somatic Cell Nuclear Transfer.
Abstract The combination of CRISPR/Cas9 and SCNT techniques greatly facilitates the production of gene-edited livestock. Here, we describe the detailed procedure to create gene knockout goats using this strategy starting from the construction of CRISPR/Cas9 targeting vectors to the transfer of cloned embryos into recipient females. In this procedure, the transfection conditions for goat fibroblasts were optimized due to their high sensitivity to electrotransfection, which enabled the isolation of single-cell colonies carrying simultaneous disruption of multiple genes for SCNT with a single co-transfection of poole...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Fan Z, Yang M, Regouski M, Polejaeva IA Tags: Methods Mol Biol Source Type: research

Production of Transgenic Chickens Using Cultured Primordial Germ Cells and Gonocytes.
Abstract The unique characteristics of the avian embryo, with its large opaque yolk, have necessitated the development of different approaches to transgenesis from those that have been successful in mammalian species. Genetic modification of birds was greatly advanced by the ability to grow long-term cultures of primordial germ cells (PGCs). These cells are obtained from embryos, established in culture, and can be propagated without losing the ability to contribute to the germline when reintroduced into a host animal. PGCs can be genetically modified in culture using traditional transfection and selection techniqu...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Collarini EJ, Leighton PA, Van de Lavoir MC Tags: Methods Mol Biol Source Type: research

Using Microinjection to Generate Genetically Modified Caenorhabditis elegans by CRISPR/Cas9 Editing.
Abstract In this chapter, we describe the procedure for generating genetically modified Caenorhabditis elegans using microinjection via the Cas9-mediated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing technique. Specifically, we describe the detailed method of performing CRISPR editing by microinjection using the Cloning-free Co-CRISPR method described by the Seydoux lab. This microinjection protocol can also be used for CRISPR editing with protocols from other labs as well as for a variety of other editing techniques including Mos1-mediated single-copy transgene insertions (MosSCI), tr...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Iyer J, DeVaul N, Hansen T, Nebenfuehr B Tags: Methods Mol Biol Source Type: research

Functional Studies of Transcriptional Cofactors via Microinjection-Mediated Gene Editing in Xenopus.
Abstract The anuran Xenopus laevis has been studied for decades as a model for vertebrate cell and developmental biology. More recently, the highly related species Xenopus tropicalis has offered the opportunity to carry out genetic studies due to its diploid genome as compared to the pseudo-tetraploid Xenopus laevis. Amphibians undergo a biphasic development: embryogenesis to produce a free-living tadpoles and subsequent metamorphosis to transform the tadpole to a frog. This second phase mimics the so-called postembryonic development in mammals when many organs/tissues mature into their adult form in the presence ...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Shibata Y, Bao L, Fu L, Shi B, Shi YB Tags: Methods Mol Biol Source Type: research

Microinjection of Live Mammalian Cells: A Delivery Method that Provides Added Versatility to the Study of Cellular Function.
Abstract Microinjection is a technique that allows for the delivery of a diverse array of compounds and biomolecules into live mammalian cells. As a result, the behavior of injected biomolecules and their resulting effects can be observed in the native environments of mammalian cells in real time. This capability allows for more accurate observations and conclusions about biological systems of interest. This chapter discusses the protocol and guidelines to successfully microinject live mammalian cells to maintain their viability. This chapter outlines considerations into the preparation of samples and the microsco...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Gahl RF Tags: Methods Mol Biol Source Type: research

Sieve Elements: The Favourite Habitat of Phytoplasmas.
Abstract The sieve elements are the only plant compartments, where phytoplasmas can survive and propagate. Therefore, this chapter is focussed on the specific molecular and cell-biological properties of the sieve element. Sieve element-companion cell complexes arise from (pro)cambial mother cells induced by key genes known to be decisive for sieve-element differentiation. The special anatomy, cell biology, and plasma-membrane outfit of sieve elements allows them to act collectively as a tube system that is able to drive a mass flow against the flow induced by transpiration. Plasmodesmal corridors are vital for the...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: van Bel AJE Tags: Methods Mol Biol Source Type: research

Characterization of Phytoplasmal Effector Protein Interaction with Proteinaceous Plant Host Targets Using Bimolecular Fluorescence Complementation (BiFC).
Abstract Elucidating the molecular mechanisms underlying plant disease development has become an important aspect of phytoplasma research in the last years. Especially unraveling the function of phytoplasma effector proteins has gained interesting insights into phytoplasma-host interaction at the molecular level. Here, we describe how to analyze and visualize the interaction of a phytoplasma effector with its proteinaceous host partner using bimolecular fluorescence complementation (BiFC) in Nicotiana benthamiana mesophyll protoplasts. The protocol comprises a description of how to isolate protoplasts from leaves ...
Source: Mol Biol Cell - October 27, 2018 Category: Molecular Biology Authors: Janik K, Stellmach H, Mittelberger C, Hause B Tags: Methods Mol Biol Source Type: research

Ex-Vivo Signal Transduction Studies in Chronic Lymphocytic Leukemia.
Abstract Microenvironmental signaling is pivotal to chronic lymphocytic leukemia (CLL) pathology; therefore understanding how to investigate this pathway by both protein and chemical methods is crucial if we are to investigate and correlate biological changes with therapeutic responses in patients. Herein, we describe the use of western blotting also referred to as immunoblotting as a method that can semiquantitatively evaluate changes in protein expression following receptor engagement; this includes B cell receptor (BCR) signaling following stimulation with anti-IgM (Blunt et al. Clin Cancer Res 23(9):2313-2324,...
Source: Mol Biol Cell - October 24, 2018 Category: Molecular Biology Authors: Rogers-Broadway KR, Karydis LI, Dobson RC, Steele AJ Tags: Methods Mol Biol Source Type: research

Culture and Harvest of CpG-Stimulated Peripheral Blood or Bone Marrow in Chronic Lymphocytic Leukemia.
Abstract Chromosome analysis of chronic lymphocytic leukemia (CLL) is an important clinical tool for evaluating prognosis and disease progression. Visualizing chromosomes microscopically using traditional cytogenetic techniques requires dividing cells to be arrested during metaphase. The major challenge for performing this analysis on CLL samples is stimulating the cells to divide in culture. Stimulation of CLL cells with CpG oligodeoxynucleotides has improved our ability to perform chromosome analysis for this leukemia. This protocol should help the reader successfully culture CLL samples for clinical chromosome ...
Source: Mol Biol Cell - October 24, 2018 Category: Molecular Biology Authors: Miller CR, Heerema NA Tags: Methods Mol Biol Source Type: research