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

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.

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

Mechanisms of Development ◽

10.1016/j.mod.2019.103558 ◽

2019 ◽

Vol 158 ◽

pp. 103558 ◽

Cited By ~ 1

Author(s):

Shuanqing Li ◽

Yunxiao Li ◽

Han Li ◽

Ciqing Yang ◽

Juntang Lin

Keyword(s):

Spinal Cord ◽

Gene Function ◽

Function Analysis ◽

Slice Culture ◽

Embryonic Spinal Cord ◽

Gene Function Analysis

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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.

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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.

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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.

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