Use of in vitro electroporation and slice culture for gene function analysis in the mouse embryonic spinal cord

Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System

Insects ◽

10.3390/insects11010012 ◽

2019 ◽

Vol 11 (1) ◽

pp. 12

Author(s):

Chenchen Lu ◽

Zhiqing Li ◽

Li Chang ◽

Zhaoming Dong ◽

Pengchao Guo ◽

...

Keyword(s):

Gene Function ◽

Culture Medium ◽

Target Genes ◽

High Efficiency ◽

Function Analysis ◽

Binding Capacity ◽

Pamam Dendrimers ◽

Binding Property ◽

Gene Function Analysis

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

Download Full-text

Efficient Regeneration of Tobacco (Nicotiana tabacum L.) Plantlets from Cotyledon, Hypocotyl and Leaf Explants: An Excellent Model Plant for Gene Function Analysis

Current Journal of Applied Science and Technology ◽

10.9734/cjast/2020/v39i3230996 ◽

2020 ◽

pp. 1-9

Author(s):

Md. Shoyeb ◽

Kanis Fatema ◽

Md. Abdur Rauf Sarkar ◽

Atikur Rahman ◽

Shaikh Mizanur Rahman

Keyword(s):

Gene Function ◽

Callus Induction ◽

Function Analysis ◽

In Vitro Regeneration ◽

Leaf Explants ◽

Leaf Explant ◽

Regeneration Ability ◽

Ms Medium ◽

Gene Function Analysis

Tobacco has been widely used as a model plant for stable and non-stable gene function analysis. Successful Agrobacterium-mediated transformation mainly depends on in vitro regeneration of tobacco plant. However, a reliable and standard regeneration protocol of tobacco using multiple explants is limited. In this study, we established a reliable and reproducible regeneration protocol of tobacco using three different explants i.e. cotyledon, hypocotyl and leaf. Preliminary, surface sterilized tobacco seeds were germinated on growth regulator free MS medium. Thereafter, in vitro germinated explants were inoculated into Murashige and Skoog [1] media supplemented with different combination and types of growth regulators for callus induction and subsequent regeneration of plantlets. It was revealed that, regeneration ability of explants is greatly influenced by type and nature of the explant. Among the three explants, higher callus induction (95%) was obtained in MS medium supplemented with 2.0 mg l-1 kinetin + 2.0 mg l-1 IAA from leaf explant. Also, leaf explant exhibited much higher regeneration ability (95%) than hypocotyl (60%) and cotyledon (45%) explants. Significantly highest number of shoots (8.0) were regenerated from leaf explants cultured on MS medium supplemented with 3.0 mg l-1 Kinetin+1.0 mg l-1 IAA compared to the other hormone combinations. Regenerated mature shoots were showed normal root after transferred onto ½ MS medium containing 0.3 mg l-1 IBA. This study will provide valuable information related to in vitro regeneration of tobacco plantlets using cotyledon, hypocotyl and leaf explants and will be used as a standard protocol for Agrobacterium-mediated transformation for gene function analysis.

Download Full-text

Gene Function Analysis in the Ubiquitous Human Commensal and PathogenMalasseziaGenus

mBio ◽

10.1128/mbio.01853-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 23

Author(s):

Giuseppe Ianiri ◽

Anna F. Averette ◽

Joanne M. Kingsbury ◽

Joseph Heitman ◽

Alexander Idnurm

Keyword(s):

Gene Function ◽

Genetic Manipulation ◽

Function Analysis ◽

Exogenous Dna ◽

Melanin Biosynthesis ◽

Disease Symptoms ◽

Wide Range ◽

Transformation Methods ◽

Gene Function Analysis

ABSTRACTThe genusMalasseziaincludes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation ofMalassezia furfurandMalassezia sympodialisusingAgrobacterium tumefaciensdelivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case ofM. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of theADE2gene for purine biosynthesis and of theLAC2gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible inMalasseziaspecies.IMPORTANCESpecies in the genusMalasseziaare a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culturein vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species ofMalassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

Download Full-text

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽

10.1186/s13007-021-00775-w ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Decai Tuo ◽

Peng Zhou ◽

Pu Yan ◽

Hongguang Cui ◽

Yang Liu ◽

...

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Gene Function ◽

Viral Vector ◽

Function Analysis ◽

Systemic Infection ◽

Cdna Clones ◽

Virus Induced Gene Silencing ◽

Efficient Gene ◽

Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Download Full-text

Apoptosis in metanephric development.

The Journal of Cell Biology ◽

10.1083/jcb.119.5.1327 ◽

1992 ◽

Vol 119 (5) ◽

pp. 1327-1333 ◽

Cited By ~ 200

Author(s):

C Koseki ◽

D Herzlinger ◽

Q al-Awqati

Keyword(s):

Spinal Cord ◽

Protein Kinase ◽

Transcriptional Activation ◽

Phorbol Esters ◽

Dna Degradation ◽

Morphological Evidence ◽

Metanephric Mesenchyme ◽

Embryonic Spinal Cord

During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin-D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation.

Download Full-text

Effects of Electroacupuncture and the Retinoid X Receptor (Rxr) Signalling Pathway on Oligodendrocyte Differentiation in the Demyelinated Spinal Cord of Rats

Acupuncture in Medicine ◽

10.1136/acupmed-2016-011134 ◽

2017 ◽

Vol 35 (2) ◽

pp. 122-132 ◽

Cited By ~ 3

Author(s):

Xiao-Hua Yang ◽

Ying Ding ◽

Wen Li ◽

Rong-Yi Zhang ◽

Jin-Lang Wu ◽

...

Keyword(s):

Spinal Cord ◽

Retinoid X Receptor ◽

Signalling Pathway ◽

Control Group ◽

Oligodendrocyte Differentiation ◽

Adult Rats ◽

Group Protein ◽

Embryonic Spinal Cord ◽

Oligodendrocyte Precursor

Objectives In spinal cord demyelination, some oligodendrocyte precursor cells (OPCs) remain in the demyelinated region but have a reduced capacity to differentiate into oligodendrocytes. This study investigated whether ‘Governor Vessel’ (GV) electroacupuncture (EA) would promote the differentiation of endogenous OPCs into oligodendrocytes by activating the retinoid X receptor γ (RXR-γ)-mediated signalling pathway. Methods Adult rats were microinjected with ethidium bromide (EB) into the T10 spinal cord to establish a model of spinal cord demyelination. EB-injected rats remained untreated (EB group, n=26) or received EA treatment (EB+EA group, n=26). A control group (n=26) was also included that underwent dural exposure without EB injection. After euthanasia at 7 days (n=5 per group), 15 days (n=8 per group) or 30 days (n=13 per group), protein expression of RXR-γ in the demyelinated spinal cord was evaluated by immunohistochemistry and Western blotting. In addition, OPCs derived from rat embryonic spinal cord were cultured in vitro, and exogenous 9-cis-RA (retinoic acid) and RXR-γ antagonist HX531 were administered to determine whether RA could activate RXR-γ and promote OPC differentiation. Results EA was found to increase the numbers of both OPCs and oligodendrocytes expressing RXR-γ and RALDH2, and promote remyelination in the remyelinated spinal cord. Exogenous 9-cis-RA enhanced the differentiation of OPCs into mature oligodendrocytes by activating RXR-γ. Conclusions The results suggest that EA may activate RXR signalling to promote the differentiation of OPCs into oligodendrocytes in spinal cord demyelination.

Download Full-text

The Organotypic Longitudinal Spinal Cord Slice Culture for Stem Cell Study

Stem Cells International ◽

10.1155/2015/471216 ◽

2015 ◽

Vol 2015 ◽

pp. 1-10 ◽

Cited By ~ 11

Author(s):

Joanna Sypecka ◽

Sylwia Koniusz ◽

Maria Kawalec ◽

Anna Sarnowska

Keyword(s):

Spinal Cord ◽

Molecular Mechanisms ◽

Synapse Formation ◽

Scientific Basis ◽

Slice Culture ◽

Spinal Cord Regeneration ◽

Spinal Cord Slice ◽

Detailed Method ◽

Cord Injury

The objective of this paper is to describe in detail the method of organotypic longitudinal spinal cord slice culture and the scientific basis for its potential utility. The technique is based on the interface method, which was described previously and thereafter was modified in our laboratory. The most important advantage of the presented model is the preservation of the intrinsic spinal cord fiber tract and the ventrodorsal polarity of the spinal cord. All the processes occurring during axonal growth, regeneration, synapse formation, and myelination could be visualized while being culturedin vitrofor up to 4-5 weeks after the slices had been isolated. Both pups and adult animals can undergo the same, equally efficient procedures when going by the protocol in question. The urgent need for an appropriatein vitromodel for spinal cord regeneration results from a greater number of clinical trials concerning regenerative medicine in the spinal cord injury and from still insufficient knowledge of the molecular mechanisms involved in the neuroreparative processes. The detailed method of organotypic longitudinal spinal cord slice culture is accompanied by examples of its application to studying biological processes to which both the CNS inhabiting and grafted cells are subjected.

Download Full-text