scholarly journals A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

2021 ◽  
Author(s):  
Roger Caothien ◽  
Charles Yu ◽  
Lucinda Tam ◽  
Robert Newman ◽  
Brian Nakao ◽  
...  
Keyword(s):  
Animal Models ◽  
Gene Targeting ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Rapid Screening ◽  
Es Cell ◽  
Screening Process ◽  
Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

2008 ◽  
Vol 20 (9) ◽  
pp. 58
Author(s):  
G. R. Kafer ◽  
SA Lehnert ◽  
P. L. Kaye ◽  
R. J. Moser
Keyword(s):  
Messenger Rna ◽  
Expression Profiles ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Histone Variants ◽  
Histone Variant ◽  
Preimplantation Embryos ◽  
Es Cell

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.

scholarly journals Increased Apoptosis and Skewed Differentiation in Mouse Embryonic Stem Cells Lacking the Histone Methyltransferase Mll2

2007 ◽  
Vol 18 (6) ◽  
pp. 2356-2366 ◽  
Author(s):  
Sandra Lubitz ◽  
Stefan Glaser ◽  
Julia Schaft ◽  
A. Francis Stewart ◽  
Konstantinos Anastassiadis
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Lineage Commitment ◽  
Es Cell ◽  
Histone 3 ◽  
Caspase Inhibition ◽  
Transcriptional Memory

Epigenetic regulation by histone methyltransferases provides transcriptional memory and inheritable propagation of gene expression patterns. Potentially, the transition from a pluripotent state to lineage commitment also includes epigenetic instructions. The histone 3 lysine 4 methyltransferase Mll2/Wbp7 is essential for embryonic development. Here, we used embryonic stem (ES) cell lines deficient for Mll2 to examine its function more accurately. Mll2−/− ES cells are viable and retain pluripotency, but they display cell proliferation defects due to an enhanced rate of apoptosis. Apoptosis was not relieved by caspase inhibition and correlated with decreased Bcl2 expression. Concordantly, Mll2 binds to the Bcl2 gene and H3K4me3levels are reduced at the binding site when Mll2 is absent. In vitro differentiation showed delays along representative pathways for all three germ layers. Although ectodermal delays were severe and mesodermal delays persisted at about three days, endodermal differentiation seemed to recover and overshoot, concomitant with prolonged Oct4 gene expression. Hence, Mll2 is not required for ES cell self-renewal or the complex changes in gene expression involved in lineage commitment, but it contributes to the coordination and timing of early differentiation decisions.

scholarly journals Herbal Extract from Codonopsis pilosula (Franch.) Nannf. Enhances Cardiogenic Differentiation and Improves the Function of Infarcted Rat Hearts

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 422
Author(s):  
Jieh-Neng Wang ◽  
Chung-Dann Kan ◽  
Lain-Tze Lee ◽  
Lynn L. H. Huang ◽  
Ya-Li Hsiao ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Herbal Extract ◽  
Egfp Expression ◽  
Herbal Extracts ◽  
Es Cell ◽  
Codonopsis Pilosula ◽  
Cardiogenic Differentiation

Background: The roots of Codonopsis pilosula (Franch.) Nannf. have been used in traditional Chinese medicine for treating cardiovascular disease. In the current study, we aimed to discover herbal extracts from C. pilosula that are capable of improving cardiac function of infarcted hearts to develop a potential therapeutic approach. Methods: A mouse embryonic stem (ES) cell-based model with an enhanced green fluorescent protein (eGFP) reporter driven by a cardiomyocyte-specific promoter, the α-myosin heavy chain, was constructed to evaluate the cardiogenic activity of herbal extracts. Then, herbal extracts from C. pilosula with cardiogenic activity based on an increase in eGFP expression during ES cell differentiation were further tested in a rat myocardial infarction model with left anterior descending artery (LAD) ligation. Cardiac function assessments were performed using echocardiography, 1, 3, and 6 weeks post LAD ligation. Results: The herbal extract 417W from C. pilosula was capable of enhancing cardiogenic differentiation in mouse ES cells in vitro. Echocardiography results in the LAD-ligated rat model revealed significant improvements in the infarcted hearts at least 6 weeks after 417W treatment that were determined based on left ventricle fractional shortening (FS), fractional area contraction (FAC), and ejection fraction (EF). Conclusions: The herbal extract 417W can enhance the cardiogenic differentiation of ES cells and improve the cardiac function of infarcted hearts.

scholarly journals Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein

2002 ◽  
Vol 11 (7) ◽  
pp. 631-635 ◽  
Author(s):  
Tatsuyuki Takada ◽  
Yutaka Suzuki ◽  
Yasushi Kondo ◽  
Nae Kadota ◽  
Kinji Kobayashi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Green Fluorescent

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

scholarly journals Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

2006 ◽  
Vol 203 (10) ◽  
pp. 2315-2327 ◽  
Author(s):  
Eugen Kolossov ◽  
Toktam Bostani ◽  
Wilhelm Roell ◽  
Martin Breitbach ◽  
Frank Pillekamp ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Heart Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Severe Heart Failure ◽  
Mouse Heart ◽  
Es Cell ◽  
Positive Effects

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (&gt;99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

scholarly journals A CRISPR toolbox for generating intersectional genetic mice for functional, molecular, and anatomical circuit mapping

2021 ◽  
Author(s):  
Savannah Lusk ◽  
Andrew McKinney ◽  
Patrick J Hunt ◽  
Paul G. Fahey ◽  
Jay Patel ◽  
...  
Keyword(s):  
Animal Models ◽  
Cell Lines ◽  
Calcium Release ◽  
Es Cells ◽  
Ovarian Follicle ◽  
Embryonic Stem ◽  
Mouse Line ◽  
Es Cell ◽  
Widespread Application ◽  
Mouse Lines

Background A full understanding of circuits and cellular mechanisms governing health and disease requires the dissection and multi-faceted study of discrete cell subtypes in developing and adult animal models. Recombinase-driven expression of transgenic response alleles represents a significant and powerful approach to delineate cell populations for functional, molecular, and anatomical study. In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, the expense and difficulty associated with production of customized intersectional mouse lines have limited widespread application to more common allele manipulations that are often commercially produced at great expense. Results We present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production. Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stem (ES) cell lines that are designed to study how functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior. As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control. We also generated a mouse line with a single recombinase responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle. Conclusions Lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

Abstract 1017: Post-translational Modification Of GATA-4 Involved In The Differentiation Of Monkey ES Cell Into Cardiac Myocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
mohsen hosseinkhani ◽  
Hossein Hosseinkhani ◽  
Ali Khademhosseini
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cardiac Myocytes ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Left Ventricular ◽  
Specific Gene ◽  
Es Cell ◽  
Post Translational Modification

Transplantation of embryonic stem (ES) cells into infracted myocardium has been shown to preserve left ventricular function in rodents. Before application of ES cell therapy in humans, however, it is critical to perform pre-clinical studies in large animals such as primates. Characteristics of cynomolgus monkey ES cells are similar with those of human ES cells, but quite different from those of mouse ES cells. Differentiation of Embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetytrans-ferases (HATs) and Histone deactylases (HDACs) govern gene expression patterns by being recruited to the target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for actylation and DNA binding of GATA-4 and its full transcription activity as well as for promotion of a transcriptionally active chromatin configuration. The role of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of monkey ES cells into cardiac myocytes remained unknown. In an ES cell model of developing embryonic bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. TSA facilitate the expression of endogenous cardiac β-myosing heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histone are involved in the differentiation of monkey ES cells into cardiac myocytes.

ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

2013 ◽  
Vol 25 (2) ◽  
pp. 446 ◽  
Author(s):  
Ruchi Sharma ◽  
Aman George ◽  
Manmohan S. Chauhan ◽  
Suresh Singla ◽  
Radhey S. Manik ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Es Cells ◽  
Single Cells ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Rock Inhibitor

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50–80. Y-27632 increased mean colony area (P < 0.05) although it did not improve their survival. It decreased OCT4 expression (P < 0.05), increased NANOG expression (P < 0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P < 0.05) and decreased that of pro-apoptotic genes BAX and BID (P < 0.05). It increased plating efficiency of single-cell suspensions of ES cells (P < 0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P < 0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1–60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

Transgenic models for study of pulmonary development and disease

1994 ◽  
Vol 267 (5) ◽  
pp. L489-L497 ◽  
Author(s):  
S. W. Glasser ◽  
T. R. Korfhagen ◽  
S. E. Wert ◽  
J. A. Whitsett
Keyword(s):  
Epithelial Cell ◽  
Animal Models ◽  
Gene Targeting ◽  
Transgenic Mouse ◽  
Embryonic Stem ◽  
Lung Epithelial Cell ◽  
Major Advance ◽  
Cis Acting ◽  
Lung Epithelial

This review summarizes progress in the application of transgenic mouse technology to the study of lung development and disease. Since advances in molecular genetics have greatly facilitated the isolation of cDNA and genes, our ability to readily assess roles of both normal and mutated genes in transgenic mouse in vivo represents a major advance, bridging molecular biology and whole animal physiology. Strategies have been developed in which lung epithelial cell promoter elements are used to drive normal or mutated genes into specific subsets of respiratory epithelial cells in the lungs of developing and mature transgenic mice. These mice have been used to elucidate the cis-acting elements controlling lung epithelial cell gene expression, to discern the role of specific polypeptides in lung morphogenesis and tumorigenesis, and to create animal models of pulmonary disease. The ability to mutate genes at their precise chromosomal locations through gene targeting in embryonic stem cells has lead to the production of animal models of lung diseases such as cystic fibrosis. Both gene insertion and gene targeting create permanent mouse lines that pass the modified gene to their progeny, providing animals for the study of the pathogenesis and treatment of pulmonary disorders.

scholarly journals Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

2019 ◽  
Vol 317 (4) ◽  
pp. C725-C736
Author(s):  
Gurbind Singh ◽  
Divya Sridharan ◽  
Mahmood Khan ◽  
Polani B. Seshagiri
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Monochromatic Light ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Medical Diagnostics ◽  
Calcium Transients ◽  
Egfp Expression ◽  
Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

Sign in / Sign up

Export Citation Format

Share Document