mRNA Transfection of Mouse and Human Neural Stem Cell Cultures
Publication Date
December 26, 2013
Journal
PLOS ONE
Authors
Samuel Mc Lenachan, Dan Zhang, Ana Belén Alvarez Palomo, Michael J. Edel, et al
Volume
8
Issue
12
Pages
e83596
DOI
https://dx.plos.org/10.1371/journal.pone.0083596
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0083596
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/24386231
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3873397
Europe PMC
http://europepmc.org/abstract/MED/24386231
Web of Science
000329116700060
Scopus
84893433251
Mendeley
http://www.mendeley.com/research/mrna-transfection-mouse-human-neural-stem-cell-cultures-2
Events
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Mendeley | Further Information

{"title"=>"mRNA transfection of mouse and human neural stem cell cultures", "type"=>"journal", "authors"=>[{"first_name"=>"Samuel", "last_name"=>"McLenachan", "scopus_author_id"=>"8601109500"}, {"first_name"=>"Dan", "last_name"=>"Zhang", "scopus_author_id"=>"57198599958"}, {"first_name"=>"Ana Belén Alvarez", "last_name"=>"Palomo", "scopus_author_id"=>"56025803200"}, {"first_name"=>"Michael J.", "last_name"=>"Edel", "scopus_author_id"=>"6701396968"}, {"first_name"=>"Fred K.", "last_name"=>"Chen", "scopus_author_id"=>"7404908114"}], "year"=>2013, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84893433251", "sgr"=>"84893433251", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0083596", "pmid"=>"24386231", "pui"=>"372294863"}, "id"=>"63af4969-e9d7-37c0-9bf1-5d9b19a8ac3e", "abstract"=>"The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.", "link"=>"http://www.mendeley.com/research/mrna-transfection-mouse-human-neural-stem-cell-cultures-2", "reader_count"=>47, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>12, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>13, "Student > Master"=>8, "Student > Bachelor"=>8, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>12, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>13, "Student > Master"=>8, "Student > Bachelor"=>8, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>1, "Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>8, "Materials Science"=>1, "Agricultural and Biological Sciences"=>22, "Medicine and Dentistry"=>6, "Neuroscience"=>5, "Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>1}, "Materials Science"=>{"Materials Science"=>1}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>6}, "Neuroscience"=>{"Neuroscience"=>5}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>22}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>8}, "Unspecified"=>{"Unspecified"=>3}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Czech Republic"=>1}, "group_count"=>0}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1328973"], "description"=>"<p><b>A:</b> Adult mouse neurospheres were transfected in 24 well plates with 500ng of EGFP mRNA using the lipofection reagents Lipofectamine 2000 or Transit or with 1 µg of EGFP mRNA using the NEON microporation system. Transfection efficiency (left panel) and EGFP fluorescence per cell (right panel) were measured by flow cytometry. 24 hours after transfection. <b>B:</b> Mouse neurospheres were cultured as either adherent monolayers (dashed line) or floating neurospheres (solid line) in 24 well plates and transfected with increasing amounts of EGFP mRNA lipoplexes. Transfection efficiency (left panel) and EGFP fluorescence per cell (right panel) were measured by flow cytometry. <b>C:</b> Whole mouse (solid line) or human (dashed line) neurospheres were transfected with increasing concentrations of mRNA using the NEON microporation system and seeded into 24 well plates. Transfection efficiency (%EGFP, dashed line) and EGFP fluorescence per cell (MPF, solid line) were measured by flow cytometry. <b>D:</b> Mouse neurospheres were transfected with 500ng of EGFP mRNA lipoplexes (solid line), pEGFP-N1 DNA lipoplexes (dotted line) or 1 µg of EGFP mRNA using the NEON microporator (dashed line). Transfection efficiency (left panel) and EGFP fluorescence per cell (right panel) were measured by flow cytometry 24, 48, 72 and 120 hours after transfection.</p>", "links"=>[], "tags"=>["developmental biology", "stem cells", "Adult stem cells", "Embryonic stem cells", "Neural stem cells", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "Cellular types", "gene expression", "Nucleic acids", "neurospheres", "egfp"], "article_id"=>886903, "categories"=>["Biological Sciences"], "users"=>["Samuel McLenachan", "Dan Zhang", "Ana Belén Alvarez Palomo", "Michael J. Edel", "Fred K. Chen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083596.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transfection_of_Mouse_Neurospheres_with_EGFP_mRNA_/886903", "title"=>"Transfection of Mouse Neurospheres with EGFP mRNA.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-26 04:13:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1328975"], "description"=>"<p><b>A:</b> Adult mouse neurospheres were cultured in 96 well plates and transfected with increasing concentrations of mRNA lipoplexes using Lipofectamine 2000 or TransIT. Two days after transfection, cell numbers were quantitated using the MTT assay. <b>B:</b> Mouse neurospheres were plated onto geltrex-coated 24 well plates 24 hours after transfection with 500ng of mRNA lipoplexes. After overnight culture, resulting monolayers were fixed and the proportion of proliferating cells determined by Ki67 immunostaining. Each bar represents the mean percentage of Ki67<sup>+</sup> cells from 6-8 transfections. Error bars indicate standard deviation. <b>C:</b> Transfected mouse neurospheres were plated as described in B and immunostained for Ki67, Nestin or βIII-Tubulin. Micrographs show merged images consisting of the antibody signal (red), EGFP fluorescence (green). Scale bars indicate 50 µm. <b>D:</b> Mouse neurospheres were transfected with 500ng of mRNA lipoplexes, plated onto geltrex and cultured without mitogens for 3 days before immunostaining for βIII-Tubulin (red) and DAPI (blue). Scale bar indicates 50 µm. <b>E:</b> Quantitation of neuronal cells. Each bar represents the mean percentages of βIII-Tubulin<sup>+</sup> cells from 3 transfections. Error bars indicate standard deviation.</p>", "links"=>[], "tags"=>["developmental biology", "stem cells", "Adult stem cells", "Embryonic stem cells", "Neural stem cells", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "Cellular types", "gene expression", "Nucleic acids", "egfp", "mrna"], "article_id"=>886905, "categories"=>["Biological Sciences"], "users"=>["Samuel McLenachan", "Dan Zhang", "Ana Belén Alvarez Palomo", "Michael J. Edel", "Fred K. Chen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083596.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Lipofection_of_EGFP_mRNA_in_Mouse_Neurospheres_/886905", "title"=>"Lipofection of EGFP mRNA in Mouse Neurospheres.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-26 04:13:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1328977"], "description"=>"<p><b>A:</b> Adult mouse neurospheres were cultured as whole NS in 24 well plates and transfected with 500ng of EGFP mRNA lipoplexes. Two days after the first transfection, a second dose of lipoplexes was applied to the cultures. Transfection efficiencies were measured by flow cytometry and fluorescence microscopy 72(left panel), a single dose (middle panel) or two doses (right panel) of mRNA lipoplexes. <b>B:</b> Total RNA was extracted adult mouse neurospheres and IFNβ expression measured by RT-PCR (lower bands) 24 hours after transfection. RT-PCR for GAPDH mRNA (upper band) was performed as a positive control. Lanes in the left panel show untransfected control neurospheres (C-), neurospheres treated with LF2000 alone (V-) or with EGFP mRNA lipoplexes (E-) after a single (24) or double (48) dose of lipoplexes. The right panel shows IFNβ expression after a single transfection of increasing amounts (0, 200ng, 500ng, 1 µg, 2 µg) of EGFP mRNA lipoplexes. The 1 kb Plus DNA Ladder (Life Technologies) is shown in the first lane of each panel.</p>", "links"=>[], "tags"=>["developmental biology", "stem cells", "Adult stem cells", "Embryonic stem cells", "Neural stem cells", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "Cellular types", "gene expression", "Nucleic acids", "transfection"], "article_id"=>886907, "categories"=>["Biological Sciences"], "users"=>["Samuel McLenachan", "Dan Zhang", "Ana Belén Alvarez Palomo", "Michael J. Edel", "Fred K. Chen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083596.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Multiple_Transfection_of_Mouse_Neurospheres_/886907", "title"=>"Multiple Transfection of Mouse Neurospheres.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-26 04:13:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1328982"], "description"=>"<p><b>A-D:</b> Human embryonic stem cell-derived neurospheres were transfected using LF2000 in suspension culture (<b>A-B</b>) or adherent monolayers (<b>C</b>) or using NEON electroporation (<b>D</b>) Micrographs were taken 24 hours after transfection. Scale bars indicate 250 µm. <b>E:</b> Human neurospheres were transfected with 500 ng of EGFP mRNA lipoplexes in 24 well plates. Two days after the first transfection, a second dose of lipoplexes was applied to the cultures. Transfection efficiencies were measured by flow cytometry and fluorescence microscopy 72 hours after the first transfection. Flow cytometry plots show neurospheres receiving no lipoplexes (left panel), a single dose (middle panel) or two doses (right panel) of mRNA lipoplexes. Insets in <b>E</b> show fluorescence micrographs of human neurospheres.</p>", "links"=>[], "tags"=>["developmental biology", "stem cells", "Adult stem cells", "Embryonic stem cells", "Neural stem cells", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "Cellular types", "gene expression", "Nucleic acids", "transfection"], "article_id"=>886912, "categories"=>["Biological Sciences"], "users"=>["Samuel McLenachan", "Dan Zhang", "Ana Belén Alvarez Palomo", "Michael J. Edel", "Fred K. Chen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083596.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_mRNA_Transfection_of_Human_Neurospheres_/886912", "title"=>"mRNA Transfection of Human Neurospheres.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-26 04:13:25"}
  • {"files"=>["https://ndownloader.figshare.com/files/1328984"], "description"=>"<p><b>A:</b> Adult mouse neurospheres were seeded as single cells and cultured for 3 or 6 days. Day 4 neurospheres and day 6 neurospheres were dissociated, fixed and stained with PI for cell cycle analysis by flow cytometry (black bars) or were plated onto geltrex and stained for Ki67 (white bars). <b>B:</b> Mouse neurospheres were transfected with 500ng EGFP mRNA on day 4 or day 5 after seeding as single cells. Transfection efficiencies were measured by flow cytometry after 24 hours. <b>C:</b> Day 3 adult mouse neurospheres were transfected with 500ng of CyclinD1 or mRNA lipoplexes (lower plot) or LF2000 alone (upper plot). 24 hours after transfection, cells were dissociated and cell cycle profiles obtained by PI staining and flow cytometry. <b>D:</b> Day 6 mouse neurospheres were transfected with 1 µg of mRNA lipoplexes containing either EGFP mRNA or EGFP mRNA plus Cyclin D1 mRNA (500ng each). Transfection efficiency was measured by flow cytometry 24 hours later.</p>", "links"=>[], "tags"=>["developmental biology", "stem cells", "Adult stem cells", "Embryonic stem cells", "Neural stem cells", "Model organisms", "Animal models", "mouse", "Molecular cell biology", "Cellular types", "gene expression", "Nucleic acids", "d1", "neurosphere"], "article_id"=>886914, "categories"=>["Biological Sciences"], "users"=>["Samuel McLenachan", "Dan Zhang", "Ana Belén Alvarez Palomo", "Michael J. Edel", "Fred K. Chen"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0083596.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cyclin_D1_enhances_mouse_neurosphere_transfection_/886914", "title"=>"Cyclin D1 enhances mouse neurosphere transfection.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-12-26 04:13:25"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"22", "full-text"=>"27", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"10"}
  • {"unique-ip"=>"12", "full-text"=>"11", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2019", "month"=>"12"}
  • {"unique-ip"=>"11", "full-text"=>"9", "pdf"=>"2", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"1", "year"=>"2020", "month"=>"2"}
  • {"unique-ip"=>"13", "full-text"=>"15", "pdf"=>"3", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"3"}
  • {"unique-ip"=>"14", "full-text"=>"12", "pdf"=>"4", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2020", "month"=>"4"}

Relative Metric

{"start_date"=>"2013-01-01T00:00:00Z", "end_date"=>"2013-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences/Biochemistry", "average_usage"=>[266, 468, 593, 703, 804, 903, 993, 1084, 1171, 1256, 1339, 1422, 1492]}, {"subject_area"=>"/Biology and life sciences/Developmental biology", "average_usage"=>[275, 472, 605, 720, 822, 921, 1013, 1106, 1200, 1289, 1378, 1459, 1531]}, {"subject_area"=>"/Biology and life sciences/Genetics", "average_usage"=>[284, 491, 620, 738, 843, 945, 1043, 1137, 1225, 1315, 1400, 1479, 1555]}]}
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