Vectors, Clone information and Methods

Adenoviral vectors       are produced using standard cloning techniques, followed by homologous recombination according to a procedure adapted from Graham and Prevec (1994). High purity viral stocks are then obtained using CsCl ultracentrifugation. This yields replication-deficient and non-toxic vectors which are capable of transgene transfer into neurones both in vivo and in vitro. Adenoviral vectors are either microinjected into the relevant brain nuclei or used to transfect organotypic slice cultures. Transgene expression is detectable from around 6h after infection, fully developed 48-72h and still taking place up to 3-4 weeks post infection.

Lentiviral vectors have been recently established in this lab via a collaboration with Prof. M. Raizada' group (University of Florida, USA). These vectors are based on the modified HIV genome. They integrate into the host chromosome and therefore are able to cause very long-term expression. For details see:

Efficient Large-Scale Production and Concentration of HIV-1-Based Lentiviral Vectors For Use In vivo

Coleman,J.E.; Huentelman,M.J.; Kasparov,S.; Metcalfe,B.L.; Paton,J.F.R.; Katovich,M.J.; Semple-Rowland,S.L.; Raizada,M.K.

Physiological Genomics (2003), 12, 221-228

CLONES

LENTIVIRAL PLASMIDS (CLICK TO DOWNLOAD, or Right-click and "save to folder". Open with Vector NTI or any other software which reads *.gb files. These files can be also open as text files to extract the sequences).

pTYF-2xGfaABC1D-tTA   - "SuperGFAP driving expression of Tet-off"

pTYF-2xSYN-tTA            -    "SyperSYN1 driving expression of Tet-off"

pTYF-2xGfaABC1D-tTA   - "SuperGFAP driving expression of Tet-off"

pTYF-mCMS-SYN-EGFP                     - (clone 238)  "Bi-ridectional SYN1 driving expression of EGFP"

pTYF-mCMV-GfaABC1D-EGFP           - (clone 248)  "Bi-directional short GFAP driving expression of EGFP"

Information about plasmids for Lentivirus production

Organotypic Brain Slices Cultures  

of the rat brain(stem) are used as the main in vitro model to be combined with viral transgenesis. They are prepared by the static culturing method, modified from Stoppini et al. Briefly, 250 micrometer thick brainstem slices are prepared from rat pups (postnatal day 8-10) by conventional methods but under sterile conditions. The slices containing the NTS (or any other part of the brain) are plated on suspended 0.4 mm pore membranes (BioporeTM CM hydrophylized PTFE; Millipore) and cultured at an interface between serum-supplemented medium and 5% CO2 at 37°C. Media are exchanged twice a week. 

Cultures also can be prepared from specific strains such as spontanously hypertensive rats SHR and WKY controls. 

At the culturing age (typically P8), hypertension is not yet established in SHR but genetic factors that predispose these rats to developing 

hypertension already impact on intracellular signalling as shown in dissociated neuronal cultures from perinatal rats.

They can be used for imaging, electrophysiological experiments, transmitter release measurements, gene profiling and other applications. For details see:

S. Kasparov, A. Teschemacher, and J. F. R. Paton. Dynamic confocal imaging in acute brain slices and organotypic slice cultures using a spectral confocal microscope with single photon excitation. Experimental Physiology 87 (6):715-724, 2002.

Viral Gene Delivery into Brainstem Nuclei in Combination with Telemetry

H. Waki, S. Kasparov, L.-F. Wong, D. Murphy, T. Shimizu, and J. F. R. Paton. Chronic inhibition of eNOS activity in NTS enhances baroreceptor reflex in conscious rats. Journal of Physiology 546 (1):233-242, 2003.

Quantitative Analysis of Gene Expression using "Real Time" PCR (Opticon)

The Opticon system can be used to accurately quantify the initial concentration of a template in a sample.

In a quantitative experiment, samples of known concentration are run, then a standard curve is generated 

by graphing the logarithm of known quantities vs. the threshold cycle for those samples.  

Quantification of unknowns is performed by determining the amplification cycle at which the measured 

fluorescence exceeds the background (i.e. the threshold cycle, or “C(t)”). 

When the C(t) is applied to the standard curve generated from the known samples, 

the initial concentration of the unknowns can be calculated to a high degree of accuracy.

For further details see this link

Read a  review about real-time PCR

Confocal Imaging of living Neurones

We are using Leica SP confocal systems based on the upright fixed-stage microscope with water immersion lenses. We have established protocols which allow to visualise various population of neurones targeted using viral vectors expressing different fluorescent proteins in the same experiment. Organotypic slice cultures provide an excellent model for this kind of approach. 

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