Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 339-344 ◽  
Author(s):  
Marcella Pecora Milazzotto ◽  
Marcelo Demarchi Goissis ◽  
Weber Beringui Feitosa ◽  
Leydson Ferreira Martins ◽  
Bryan Eric Strauss ◽  
...  
Keyword(s):  
Muscle Mass ◽  
Lentiviral Vector ◽  
C2c12 Cell ◽  
Negative Regulator ◽  
C2c12 Cells ◽  
Gene Knockdown ◽  
Epifluorescence Microscopy ◽  
Bovine Embryos ◽  
Myostatin Gene

SummaryMyostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.

4 MYOSTATIN GENE KNOCKDOWN THROUGH LENTIVIRAL VECTOR-MEDIATED DELIVERY OF shRNA FOR IN VITRO PRODUCTION OF TRANSGENIC BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 120 ◽  
Author(s):  
M. P. Milazzotto ◽  
W. B. Feitosa ◽  
B. E. Strauss ◽  
M. Bajgelman ◽  
C. M. Mendes ◽  
...  
Keyword(s):  
Real Time ◽  
Cell Morphology ◽  
Real Time Pcr ◽  
Lentiviral Vector ◽  
Gene Knockdown ◽  
Control Group ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Transgenic Embryo

The main goal of husbandry and beef cattle production is to enhance performance rates, for example, weight gain. Myostatin is referred to as a negative regulator of skeletal muscle growth. Genetic engineering of this character in order to produce double muscling animals that can transmit to future progeny will enhance its usefulness. The present research aimed to analyze myostatin inhibition through lentiviral-mediated delivery of shRNA in mouse myoblast culture and the feasibility of the lentiviral-mediated delivery of shRNA into in vitro-produced transgenic bovine embryos. In order to achieve knockdown of myostatin in cell and embryo culture, a lentiviral vector was constructed with ubiquitin C promoter-driven GFP gene (green fluorescent protein) and shRNA to suppress myostatin gene expression driven by the U6 promoter. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR of myostatin and GAPDH genes. Later, bovine oocytes were in vitro-matured and the lentiviral vector was microinjected into the oocyte perivitelline space (2.5 � 106 IU mL-1) after mechanical and chemical cumulus cell removal. Non-microinjected mature oocytes were considered as control. After microinjection, oocytes were fertilized and cultured in vitro. After 4 and 9 days of culture, embryos were evaluated by epifluorescence microscopy. The GFP-positive embryos were green under fluorescence. Cell morphology and embryo development rate data were analyzed by Minitab Release 14 Statistical Software (Minitab, Inc., State College, PA, USA), submitted to ANOVA, and compared by Tukey test (P d 0.05). Real-time PCR data were analyzed by Pair-Wise Fixed Reallocation Randomization Test using REST2005 software. Cell morphology results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells as the transducted group progressed less to myotubes than in the control group. A lower amount of myostatin mRNA after 72 h of differentiation indicated an inhibition tendency by real-time PCR. In relation to the transgenic embryo production, 96.9 � 0.34% (62.65) developed to cleavage, 80.24 � 4.38% (51/65) were GFP-positive, and 50.95 � 3.37% (26/65) achieved blastocyst stage. After hatching, 3.07% (2/65) of GFP-positive embryos maintained fluorescence. In relation to the control group, the cleavage rate was 93.81 � 0.68% (61/65); the blastocyst rate 38.34 � 2.36% (25/65), and none were fluorescent. In conclusion, myostatin gene knockdown was effectively performed by lentiviral vector-mediated delivery of shRNA. Thus, novel studies about the efficiency of this vector on transgenic embryo production can be performed. This work was supported financially by FAPESP 03/0156-9.

207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM

2017 ◽  
Vol 29 (1) ◽  
pp. 212
Author(s):  
C. A. Pinzon ◽  
M. Snyder ◽  
J. Pryor ◽  
B. Thompson ◽  
M. Golding ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Muscle Mass ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Negative Regulator ◽  
Bovine Embryos ◽  
Myostatin Gene ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

The myostatin gene or growth differentiation factor 8 is a member of the transforming growth factor-β superfamily that acts as a negative regulator of muscle growth. Mutations inactivating this gene occur naturally in Piedmontese and Belgian Blue cattle breeds, resulting in a dramatic increase in muscle mass, albeit with unwanted consequences of increased dystocia and decreased fertility. Modulation of muscle mass increase without the unwanted effects would be of great value for improving livestock growth and economic value of livestock. The objective of our work was to use the CRISPR-Cas9 genetic engineering tool to generate deletions of different elements in the myostatin promoter in order to decrease the level of expression and obtain an attenuated phenotype without the detrimental consequences of an inactivating mutation. To achieve this objective 4 different small guide RNA (sgRNA) targeting the promoter near the mutation were designed with PAM positions from transcription starting site of −1577, −689, −555, and −116. These sgRNA were cloned individually into the Cas9 plasmids (px461, and px462; Addgene®). These plasmids allow for a dual puromycin resistance (px462) and green fluorescent protein (px461) selection. We first tested the functionality of these sgRNA in vitro by co-transfecting bovine fetal fibroblasts with a combination of both plasmids (Set 1 = sgRNA 1–4; Set 2 = sgRNA 2–3). Cells were exposed to puromycin (0.2 µg mL−1) for 72 h, then single and mixed colonies positive for green fluorescent protein expression were separated for propagation. The DNA was extracted for PCR amplification of the targeted region. Multiple deletions and a few insertion events were observed after PCR, bands were cloned into TOPO® vector (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced. Sequencing results confirmed the PCR products as insertions or deletions in the myostatin promoter region. We proceeded to modify the myostatin promoter directly in bovine zygotes. For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50 ng µL−1 of total sgRNA, 10 ng µL−1 of Cas9 mRNA, and 30 ng µL−1 of Cas9 protein with 1 mg mL−1 of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

Gene Expression of C2C12 Cells on Calcium Phosphate Ceramic In Vitro

2005 ◽  
Vol 288-289 ◽  
pp. 265-268 ◽  
Author(s):  
Yan Fei Tan ◽  
Ling Li Zhang ◽  
Xin Lai He ◽  
Wei Qiang Xiao ◽  
Hong Song Fan ◽  
...  
Keyword(s):  
Calcium Phosphate ◽  
Type I Collagen ◽  
C2c12 Cell ◽  
C2c12 Cells ◽  
Type I ◽  
Calcium Phosphate Ceramic ◽  
Mtt Method ◽  
Difference Expression

The osteoinduction of Calcium Phosphate (CaP) had been proved and generally been investigated by in vivo implantation. However, the mechanism of the osteoinductivity was not clear and it was difficult to judge the osteoinductivity in vitro. In this study, Mouse C2C12 cell line, a kind of myoblast precursor cell, was employed to co-culture with CaP. The induction of cell differentiation by materials was tested by MTT method, fluorescence observation, especially the mRNA expression of Osteocalcin, Type I collagen and Fibronectin by RT-PCR. It was founded that C2C12 cells could be induced to expression osteocalcin when growth on the surface of the HA/TCP ceramics. At the same time, the ceramics with different composition and sintering temperature seemed to induce difference expression level of the related genes. The results proved that phase composition was one of the most important factors in the regulation of bone-related genes. This study provided a potential model to evaluate the osteoinductivity of CaP ceramics in vitro.

Molecular analysis of fiber type-specific expression of murine myostatin promoter

2004 ◽  
Vol 287 (4) ◽  
pp. C1031-C1040 ◽  
Author(s):  
Mônica Senna Salerno ◽  
Mark Thomas ◽  
Davanea Forbes ◽  
Trevor Watson ◽  
Ravi Kambadur ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fiber Type ◽  
Muscle Growth ◽  
Quadriceps Muscle ◽  
Negative Regulator ◽  
Specific Expression ◽  
Myostatin Gene ◽  
E Boxes

Myostatin is a negative regulator of muscle growth, and absence of the functional myostatin protein leads to the heavy muscle phenotype in both mouse and cattle. Although the role of myostatin in controlling muscle mass is established, little is known of the mechanisms regulating the expression of the myostatin gene. In this study, we have characterized the murine myostatin promoter in vivo. Various constructs of the murine myostatin promoter were injected into the quadriceps muscle of mice, and the reporter luciferase activity was analyzed. The results indicate that of the seven E-boxes present in the 2.5-kb fragment of the murine myostatin promoter, the E5 E-box plays an important role in the regulation of promoter activity in vivo. Furthermore, the in vitro studies demonstrated that MyoD preferentially binds and upregulates the murine myostatin promoter activity. We also analyzed the activity of the bovine and murine promoters in murine skeletal muscle and showed that, despite displaying comparable levels of activity in murine myoblast cultures, bovine myostatin promoter activity is much weaker than murine myostatin promoter in mice. Finally, we demonstrate that in vivo, the 2.5-kb region of the murine myostatin promoter is sufficient to drive the activity of the reporter gene in a fiber type-specific manner.

scholarly journals Construction of a rAAV-SaCas9 system expressing eGFP and its application to improve muscle mass

2021 ◽  
Author(s):  
Shaoting Weng ◽  
Yitian Zhao ◽  
Changhong Yu ◽  
Xiaofan Wang ◽  
Xuehan Xiao ◽  
...  
Keyword(s):  
Muscle Mass ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Specific Site ◽  
Target Tissue ◽  
Myostatin Gene ◽  
Editing Efficiency ◽  
Green Fluorescent

AbstractAn ideal rAAV gene editing system not only effectively edits genes at specific site, but also prevents the spread of the virus from occurring off-target or carcinogenic risks. This is important for gene editing research at specific site in vivo. We report a single rAAV containing SaCas9 and guide RNAs under the control of subtle EF1a and tRNA promoters. The capacity of rAAV was compressed, and the editing efficiency was similar to that of the classical Cas9 system in vitro and in vivo. And we inserted the sequence of the green fluorescent protein eGFP into rAAV. The number of cells infected with the rAAV and the region in which the rAAV spreads were known by the fluorescent expression of eGFP in cells. In addition, we demonstrated that myostatin gene in the thigh muscles of C57BL/10 mice was knocked out by the rAAV9-SaCas9 system to make muscle mass increased obviously. The protein eGFP into rAAV has significant implications for our indirect analysis of the editing efficiency of SaCas9 in the genome of the target tissue and reduces the harm caused by off-target editing and prevents other tissue mutations. The rAAV system has substantial potential in improving muscle mass and preventing muscle atrophy.

Myostatin silencing effect on basic helix–loop–helix transcription factors in caprine foetal fibroblast cells

2017 ◽  
Author(s):  
Biswajyoti Borah ◽  
Ajit Pratap Singh ◽  
Hamen Gogoi ◽  
Amlan Jyoti Phukan ◽  
Bikash Chandra Sarkhel
Keyword(s):  
Transcription Factors ◽  
Muscle Development ◽  
Pearson Correlation ◽  
Negative Regulator ◽  
Fibroblast Cells ◽  
Animal Cells ◽  
Myostatin Gene ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

Transgenic food animal production is one of the potential and need oriented research to mitigate the food crises of the world. In vitro gene silenced animal cells and making use of these cells for transgenesis one of the suitable way to produce productive animals. Myostatin is a negative regulator of muscle growth, has the potential to increase the muscle mass upon its silencing. Four Hush 29-mer anti- myostatin (MSTN) shRNA constructs were checked for myostatin gene silencing in caprine foetal fibroblast cells and its subsequent effect on basic helix– loop–helix (bHLH) transcription factors. These factors are necessary for the terminal differentiation, proliferation, and homeostasis of muscle development. Different shRNA constructs displayed 55.1 to 91.5% (p less than 0.01) of myostatin silencing in caprine foetal fibroblast cells and upregulation of myogenic gene. Upregulation of 7.97 to 111.67 % for MyoD, 77.0 % to 319.47 % for myogenin, 16.67 % to 138.0 % for Myf5 were observed . The Pearson correlation established a negative correlation between myostatin and genes under study. Result suggests that knockdown of MSTN a potential approach to improve caprine musculatures.

scholarly journals p27Kip1 Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

2005 ◽  
Vol 16 (3) ◽  
pp. 1469-1480 ◽  
Author(s):  
Graziella Messina ◽  
Cristiana Blasi ◽  
Severina Anna La Rocca ◽  
Monica Pompili ◽  
Attilio Calconi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Myogenic Differentiation ◽  
Cell Cycle Control ◽  
C2c12 Cell ◽  
Terminal Differentiation ◽  
Active Role ◽  
C2c12 Cells ◽  
Low Density

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27Kip1 and MyoD in proliferating myoblasts. We show that inhibition of p27Kip1 expression impairs C2C12 cell differentiation at high density, while exogenous p27Kip1 allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27Kip1 is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27Kip1 in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”.

MicroRNA-139-5p regulates C2C12 cell myogenesis through blocking Wnt/β-catenin signaling pathway

2015 ◽  
Vol 93 (1) ◽  
pp. 8-15 ◽  
Author(s):  
Lin Mi ◽  
Youlei Li ◽  
Qiangling Zhang ◽  
Chen Zhao ◽  
Ying Peng ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Molecular Mechanisms ◽  
Glycogen Synthase ◽  
C2c12 Cell ◽  
Negative Regulator ◽  
C2c12 Cells ◽  
Myoblast Differentiation ◽  
C2c12 Myoblast ◽  
Myoblast Proliferation ◽  
Gsk 3Β

MicroRNAs (miRNAs) are novel and potent regulators in myogenesis. However, the molecular mechanisms that many miRNAs regulate myoblast proliferation and differentiation which are largely unknown. Here, we found that miR-139-5p increased during C2C12 myoblast proliferation, while presenting an inverse trend during C2C12 myoblast differentiation. Flow cytometry and EdU incorporation assay showed that miR-139-5p slowed down the growth of C2C12 cells. Additional study demonstrated that ectopic introduction of miR-139-5p into C2C12 cells blocked myoblast differentiation. Importantly, we demonstrated for the first time that Wnt1, which is associated with the Wnt/β-catenin signaling pathway, was a direct target of miR-139-5p. Moreover, we found that the expression level of Wnt1 was suppressed significantly (p < 0.01) by miR-139-5p, which triggered inhibition of Wnt/β-catenin signaling through upregulation of glycogen synthase kinase 3 beta (GSK-3β; p < 0.05) and downregulation of p-GSK-3β (p < 0.01), β-catenin (p < 0.05), and nuclear β-catenin (p < 0.01). Taken together, these results suggest that miR-139-5p is an important negative regulator in myogenesis through blocking the Wnt1-mediated Wnt/β-catenin signaling pathway.

scholarly journals A Three-Dimensional Culture Model of Reversibly Quiescent Myogenic Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Salvatore Aguanno ◽  
Claudia Petrelli ◽  
Sara Di Siena ◽  
Luciana De Angelis ◽  
Manuela Pellegrini ◽  
...  
Keyword(s):  
Stem Cells ◽  
C2c12 Cell ◽  
Three Dimensional ◽  
Notch Pathway ◽  
C2c12 Cells ◽  
Quiescent State ◽  
Cellular Interactions ◽  
Culture Model

Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.

scholarly journals Myostatin Gene as a Genetic Marker for Growth and Carcass Traits in Beef Cattle

2021 ◽  
Vol 31 (1) ◽  
pp. 37
Author(s):  
Peni Wahyu Prihandini ◽  
D N H Hariyono ◽  
Y A Tribudi
Keyword(s):  
Beef Cattle ◽  
Muscle Mass ◽  
Gene Polymorphisms ◽  
Carcass Traits ◽  
Phenotypic Expression ◽  
Negative Regulator ◽  
Myostatin Gene ◽  
Mstn Gene ◽  
Growth Performances ◽  
Superior Genotype

<p>Growth and carcass traits are of economic importances in livestock breeding, because they affect the profitability of animal production. The phenotypic expression of these traits is controlled by multiple genes (polygenes), such as myostatin (MSTN) gene. This paper aims to discuss the expression, polymorphism and potential application of MSTN gene as a marker-assisted selection (MAS) for growth and carcass traits in beef cattle based on data from published studies. MSTN gene or known as growth and differentiation factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) superfamily, which acts as a negative regulator of skeletal muscle mass deposition. Several published studies showed that mutations in the MSTN gene can inhibit the activation of myostatin, which leads to an increased muscle mass (hypertrophy). Several <em>MSTN</em> gene polymorphisms were reported to be associated with growth and carcass traits in local cattle in several countries, including Indonesia, namely Bali cattle. Based on several assumptions: 1) there is MTSN gene polymorphisms in a population, 2) there is a significant association between MSTN gene polymorphisms and growth and carcass traits, as reported in several beef cattle populations and 3) those cattle with superior genotype have better growth performances, we expect that there will be improvement in growth performances in the future if those cattle are selected. Understanding MSTN gene polymorphisms would be useful to make strategies for the genetic improvement for growth and carcass traits of local cattle.</p>

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