scholarly journals Gene Editing in Prunus Spp.: The Challenge of Adapting Regular Gene Transfer Procedures for Precision Breeding

2021 ◽  
Author(s):  
Ricardo Vergara ◽  
Felipe Olivares ◽  
Blanca Olmedo ◽  
Carolina Toro ◽  
Marisol Muñoz ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Gene Editing ◽  
Analysis Tool ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Enhanced Expression ◽  
Yellow Dwarf Virus ◽  
Transfer Procedures ◽  
Prunus Spp

Successfully gene editing (GE) in Prunus spp. has been delayed due to its woody nature presenting additional difficulties in both, proper regeneration protocols and designing efficient gene transfer techniques. The availability of adequate, single cell culture techniques for GE such as protoplast regeneration, is a limiting step for the genus and for this reason, the improvement of regular regeneration protocols and finding more efficient techniques for the delivery of the “editing reagents” seem to be a reasonable strategy to incorporate GE in the genus. During the last 10 years, we have focused our efforts optimizing some previous regeneration and gene transfer procedures for Japanese plum (P. salicina), sweet cherry (P. avium) and peach (P. persica) to incorporate them into a GE technology on these species. In parallel, delivery techniques for the CRISPR/Cas9 editing components, i.e., guide RNA (gRNA) and Cas9, have been developed with the aim of improving gene targeting efficiencies. In that line, using DNA virus-based replicons provides a significant improvement, as their replicational release from their carriers enables their enhanced expression. Here, we make a brief overview of the tissue culture and regeneration protocols we have developed for P. salicina, P. avium and P. persica, and then we proceed to describe the use of Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the editing reagents in vivo and to evaluate their editing capability on individuals derived from Agrobacterium-mediated gene transfer experiments of these species. We show part of our characterization assays using new BeYDV-derived vectors harboring multiple gRNAs, the Cas9 gene, and the green fluorescent protein reporter gene. We also describe a dedicated genome analysis tool, by which gRNA pairs can be designed to address gene deletions of the target genes and to predict off-target sequences. Finally, as an example, we show the general results describing GE of the peach TERMINAL FLOWER 1 gene and some preliminary characterizations of these materials.

scholarly journals CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons

2021 ◽  
Vol 12 ◽  
Author(s):  
Felipe Olivares ◽  
Rodrigo Loyola ◽  
Blanca Olmedo ◽  
María de los Ángeles Miccono ◽  
Carlos Aguirre ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Vitis Vinifera ◽  
Somatic Embryos ◽  
Gene Editing ◽  
Fluorescent Protein ◽  
Vitis Vinifera L ◽  
Thompson Seedless ◽  
Efficient Gene ◽  
Yellow Dwarf Virus

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

A T.E.M. study of mouse mammary epithelial cell secretory differentiation cultured on collagen and Matrigel

1991 ◽  
Vol 49 ◽  
pp. 188-189
Author(s):  
W.N. Bentham ◽  
V. Rocha
Keyword(s):  
Cell Culture ◽  
Mammary Epithelial ◽  
Secretory Process ◽  
Cell Culture System ◽  
Protein Maturation ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Mouse Mammary Epithelial Cell ◽  
Secretory Differentiation

It has been an interest of our lab to develop a mammary epethelial cell culture system that faithfully duplicates the in vivo condition of the lactating gland. Since the introduction of collagen as a matrix on which cells are cultivated other E.C.M. type matrices have been made available and are used in many cell culture techniques. We have previously demonstrated that cells cultured on collagen and Matrigel do not differentiate as they do in vivo. It seems that these cultures often produce cells that show a disruption in the secretory process. The appearance of large ribosomal studded vesicles, that specifically label with antibody to casein, suggest an interruption of both protein maturation and secretion at the E.R. to golgi transition. In this report we have examined cultures on collagen and Matrigel at relative high and low seeding densities and compared them to cells from the in vivo condition.

Cell Culture Methods to Evaluate the Biocompatibility of Implant Materials

1992 ◽  
Vol 20 (1) ◽  
pp. 52-60
Author(s):  
Gabriela Ciapetti ◽  
Elisabetta Cenni ◽  
Daniela Cavedagna ◽  
Loredana Pratelli ◽  
Arturo Pizzoferrato
Keyword(s):  
Cell Culture ◽  
Quantitative Data ◽  
Cell Function ◽  
Culture Methods ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Biological Compatibility ◽  
Compatibility Of Materials ◽  
Cell Culture Methods

Cell culture techniques are usually used in the field of biomaterials research and development in order to detect toxic components. Morphological assays are the most widely used methods and give the very first information about the biological compatibility of materials. Cell function assays give more quantitative data, but the comparison of data between different laboratories is difficult. Some of the cell culture methods that are used for biocompatibility studies are described briefly here, and results from our laboratory are reported. Despite some inherent limitations of the cell culture techniques, they are an accurate and reliable method of predicting the biological compatibility of materials to be implanted in vivo.

scholarly journals Microtissues in Cardiovascular Medicine: Regenerative Potential Based on a 3D Microenvironment

2016 ◽  
Vol 2016 ◽  
pp. 1-20 ◽  
Author(s):  
Julia Günter ◽  
Petra Wolint ◽  
Annina Bopp ◽  
Julia Steiger ◽  
Elena Cambria ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Retention Rate ◽  
3D Cell Culture ◽  
Traditional Role ◽  
Cell Culture Techniques ◽  
Cell Therapies ◽  
Culture Techniques ◽  
3D Microenvironment

More people die annually from cardiovascular diseases than from any other cause. In particular, patients who suffer from myocardial infarction may be affected by ongoing adverse remodeling processes of the heart that may ultimately lead to heart failure. The introduction of stem and progenitor cell-based applications has raised substantial hope for reversing these processes and inducing cardiac regeneration. However, current stem cell therapies using single-cell suspensions have failed to demonstrate long-lasting efficacy due to the overall low retention rate after cell delivery to the myocardium. To overcome this obstacle, the concept of 3D cell culture techniques has been proposed to enhance therapeutic efficacy and cell engraftment based on the simulation of an in vivo-like microenvironment. Of great interest is the use of so-called microtissues or spheroids, which have evolved from their traditional role as in vitro models to their novel role as therapeutic agents. This review will provide an overview of the therapeutic potential of microtissues by addressing primarily cardiovascular regeneration. It will accentuate their advantages compared to other regenerative approaches and summarize the methods for generating clinically applicable microtissues. In addition, this review will illustrate the unique properties of the microenvironment within microtissues that makes them a promising next-generation therapeutic approach.

scholarly journals A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1882
Author(s):  
Giovana Acha ◽  
Ricardo Vergara ◽  
Marisol Muñoz ◽  
Roxana Mora ◽  
Carlos Aguirre ◽  
...  
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Solanum Tuberosum L ◽  
Marker Gene ◽  
Leaf Explants ◽  
Analysis Tool ◽  
Targeted Deletion ◽  
Potato Genome ◽  
Guide Rna ◽  
Yellow Dwarf Virus

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on ‘Yagana-INIA’, a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

scholarly journals A cut above the rest: A new tool for microengineering cell and tissue cultures

2002 ◽  
Vol 24 (5) ◽  
pp. 25-28
Keyword(s):  
Biochemical Analysis ◽  
Cell Types ◽  
Cell Interactions ◽  
Tissue Cultures ◽  
Living Tissue ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Geometric Patterns ◽  
Distinct Cell

For biochemical analysis, the fresher the material to be analysed is, the better. With today's advanced cell-culture techniques there are a wealth of opportunities for studying biochemical pathways in fresh and even living tissue. While culture systems strive to replicate complex in vivo micro-environments, there are some aspects that remain technically challenging when using traditional methods. Controlling the ratio between distinct cell types or mimicking the geometric patterns of cell interactions found in native tissue are particular problems. Yet these aspects have been shown to be important in regulating cell–cell interactions, differentiated function and patterns of gene expression.

scholarly journals A method to culture human alveolar rhabdomyosarcoma cell lines as rhabdospheres demonstrates an enrichment in stemness and notch signaling

Biology Open ◽  
2020 ◽  
pp. bio.050211
Author(s):  
Katherine K. Slemmons ◽  
Michael D. Deel ◽  
Yi-Tzu Lin ◽  
Kristianne M. Oristian ◽  
Nina Kuprasertkul ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Cell Lines ◽  
Three Dimensional ◽  
Notch Signaling Pathway ◽  
Stem Cell Markers ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Practical Tool

The development of three-dimensional cell culture techniques has allowed cancer researchers to study the stemness properties of cancer cells in in vitro culture. However, a method to grow PAX3-FOXO1 fusion-positive rhabdomyosarcoma (FP-RMS) - an aggressive soft tissue sarcoma of childhood - has to date not been reported, hampering efforts to identify the dysregulated signaling pathways that underlie FP-RMS stemness. Here, we first examine the expression of canonical stem cell markers in human RMS tumors and cell lines. We then describe a method to grow FP-RMS cell lines as rhabdospheres and demonstrate that these spheres are enriched in expression of canonical stemness factors as well as Notch signaling components. Specifically, FP-RMS rhabdospheres have increased expression of SOX2, POU5F1 (OCT4), and NANOG, and several receptors and transcriptional regulators in the Notch signaling pathway. FP-RMS rhabdospheres also exhibit functional stemness characteristics including multipotency, increased tumorigenicity in vivo, and chemoresistance. This method provides a novel practical tool to support research into FP-RMS stemness and chemoresistance signaling mechanisms.

scholarly journals Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology

Micromachines ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 124
Author(s):  
Csaba Forro ◽  
Davide Caron ◽  
Gian Angotzi ◽  
Vincenzo Gallo ◽  
Luca Berdondini ◽  
...  
Keyword(s):  
Cell Biology ◽  
Brain Activity ◽  
Pharmaceutical Research ◽  
Brain Structures ◽  
Cell Culture Techniques ◽  
Lab On Chip ◽  
Culture Techniques ◽  
On Chip

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC–electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

scholarly journals An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

2011 ◽  
Vol 78 (1) ◽  
Author(s):  
Estelle H. Venter ◽  
Truuske Gerdes ◽  
Isabel Wright ◽  
Johan Terblanche
Keyword(s):  
Cell Culture ◽  
Bluetongue Virus ◽  
Virus Transmission ◽  
Infected Sheep ◽  
Economic Losses ◽  
Bovine Embryos ◽  
Cell Culture Techniques ◽  
Culture Techniques

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

scholarly journals Applications of novel bioreactor technology to enhance the viability and function of cultured cells and tissues

2020 ◽  
Vol 10 (2) ◽  
pp. 20190090 ◽  
Author(s):  
H. W. Hoyle ◽  
L. A. Smith ◽  
R. J. Williams ◽  
S. A. Przyborski
Keyword(s):  
Cultured Cells ◽  
Ex Vivo ◽  
Low Cost ◽  
Tissue Slices ◽  
Cell Culture Techniques ◽  
Culture Techniques ◽  
Tissue Constructs ◽  
And Function

As the field of tissue engineering continues to advance rapidly, so too does the complexity of cell culture techniques used to generate in vitro tissue constructs, with the overall aim of mimicking the in vivo microenvironment. This complexity typically comes at a cost with regards to the size of the equipment required and associated expenses. We have developed a small, low-cost bioreactor system which overcomes some of the issues of typical bioreactor systems while retaining a suitable scale for the formation of complex tissues. Herein, we have tested this system with three cell populations/tissues: the culture of hepatocellular carcinoma cells, where an improved structure and basic metabolic function is seen; the culture of human pluripotent stem cells, in which the cultures can form more heterogeneous tissues resembling the in vivo teratoma and ex vivo liver tissue slices, in which improved maintenance of cellular viability is seen over the 3 days tested. This system has the flexibility to be used for a variety of further uses and has the potential to provide a more accessible alternative to current bioreactor technologies.

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