i-Fect Deliver Plasmids to the CNS

Important for Gene Expression Studies.
I have posted many examples of how our customers use i-Fect^TM  and other Transfection Solutions for Gene Manipulation Studies. There are also many publications.

Here we feature how i-Fect was used to delivery plasmids to the CNS: Sara Elramah, María José López-González, Matthieu Bastide, Florence Dixmérias, Olivier Roca-Lapirot, Anne-Cécile Wielanek-Bachelet, Anne Vital, Thierry Leste-Lasserre, Alexandre Brochard, Marc Landry & Alexandre Favereaux. Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain. Scientific Reports 7, Article number: 10949 (2017) doi:10.1038/s41598-017-10224-1...Intrathecal administration of miRNAs and ShRNA To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments...

Figures: (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively.

I am confident there will be many more positive reports regarding our Transfection Reagents.

pn-Fect and Transfection of DRG cultures

In Vitro Gene expression analysis assays are essential for understanding how up or down regulation of  related target proteins could result in pathologies. Dorsal Root Ganglion (DRG), Neuronal and Glial Cultures have proven hard to transfect as as many transfection reagents are toxic to these cells. It is important for the study of neuro-diseases that researchers have tools and methods that enable success.

In this study, researchers successfully transfect DRG cultures with IKAP-shRNA using our pn-Fect kit. The own regulation of IKAP in these cultures support findings that helped explain the potential pathology of Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III): Hunnicutt BJ , Chaverra M , George L , Lefcort F , 2012 IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration. PLoS ONE 7(2): e32050. doi:10.1371/journal.pone.0032050.

Cell culture: Dorsal root ganglia were dissected from Embryonic day 5–9.5 chick embryos (E5–9.5) and dissociated by incubation in 0.25% trypsin-EDTA (Gibco) for 7 min at 37 C followed by trituration through fire–pulled glass pipettes. The culture media consisted of Neurobasal medium (Invitrogen) supplemented with B27 (1X,Invitrogen), Glutamax (1X,Invitrogen), Hybrimax Antibiotic\Antimicotic (1:100, Sigma), NGF (10 ng/ml, gift from Dr. Thomas Large). Cells were plated on 8-well Nunc glass chamber slides that were coated with poly-D-lysine (1:100, Sigma) and laminin 20 ug/ml (Gibco). Approximately equal numbers of cells (52,500) were plated per well. Immediately after plating, cells were transfected with IKBKAP-7.4 shRNA or control shRNA via pn-Fect (Neuromics, PN3375). Several ratios of pnfect:DNA were tested with the optimum obtained 1.84:1. The cells were then cultured for approximately 29 h at 37 C, 5.5% C02. After incubation culture cells were fixed and inmunostained as previously described [24]. To determine whether IKBKAP shRNAs altered cell proliferation and/or neuronal differentiation in dissociated DRG cultures, Brdu was added to the cultures and the cells were fixed 24 hrs later (as described in [24]). The number of GFP+/BrdU+ or GFP+/Tuj-1+ cells were quantified for each experiment, and a ratio comparing control vs. IKBKAP shRNAs for each experiment determined. For the BrdU+ experiment, a total of 556 GFP+/control shRNA transfected cells were counted and 357 GFP+/IKBKAP shRNA transfected cells in 3 separate experiment. For determining neuronal differentiation, a total of 2866 GFP+/control shRNA transfected cells and 2913 IKBKAP shRNA transfected cells were counted, over 3 separate experiments.



Images: IKAP regulates neuronal differentiation in the DRG. Reduction in IKAP leads to increased numbers of neurons in the immature DRG (A–C). Embryos at St. 12 were transfected with either control shRNAs or IKBKAP shRNAs and analyzed at St 24/25. Embryos were sectioned, and immunolabled with the neuronal markers Tuj-1 or Ben and the percentage of GFP+ neurons determined. Significantly more IKBKAP shRNA transfected DRG precursor cells differentiated into neurons (arrows in B; IKBKAP shRNA 7.4; n = 3 embryos; p = 0.002; IKBKAP shRNA 1.6 & 4.5, n = 3 embryos; p = 0.001) than Control shRNA transfected DRG precursor cells (n = 5 embryos). (D–H) IKBKAP shRNA-transfected DRG precursor cells (n = 3 embryos; 252 cleaved-Caspase 3+ cells counted) were also more likely to die by apoptosis (compare D & E to F & G) than control shRNA-electroporated cells (n = 3 embryos; 117 cleaved caspase-3+ cells counted). (H) The number of cleaved Caspase 3+ cells was quantified in DRG on both the transfected side of the embryo and the non-transfected side of the embryo and a ratio determined. Significantly more cleaved-Caspase 3+ cells were present in the transfected DRG of IKBKAP shRNA transfected embryos than in the transfected DRG in embryos transfected with control shRNAs; p = 0.006. (I–N). Over-expression of the c-terminus of IKAP prevents neural crest cells from coalescing with the DRG (I, K, L; p = 0.004) but the few that do join, tend not to differentiate into neurons (p = 0.007; J, M, N). Embryos were transfected with either a construct driving expression of the c-terminus of chicken IKAP with a His tag (CT-IKAP-His; L,N) or a control, His-tagged construct (K, M) and analyzed at St. 21. The location and neuronal identity of transfected cells was determined in 3 embryos for each treatment; Control His-plasmid: n = 343 transfected cells counted; CT-IKAP-His: n = 278 transfected cells counted. Statistical analysis by Student t-test. doi:10.1371/journal.pone.0032050.g006. 


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