Live Animal Imaging System

Xenogen IVIS 200

Location: Second floor, Central Animal Facility

Ashok M Raichur (Materials Engineering) and Saumitra Das (MCB)

Hepatocyte targeted gene delivery using asialoglycoprotein-galactose modified Chitosan-Dextran sulfate nanocapsules (CS-DS NCp) in Balb/c mice through tail vein injection was achieved. Results indicated higher percentage count of Fluc expression at mice hepatocyte. It is concluded that ASOR and ASOR-Gal modified NCp were promising candidates for hepatocyte targeted gene delivery and further it can be successfully used to deliver therapeutic molecules to address liver diseases (Figure 1).

 

Figure 1. Study of Fluc expression rate at hepatocyte. pCNDA-Fluc loaded PEI, NCp and NCp-ASOR were injected into Balb/c mice and imaged at d4 h intervals. In Day 3 mice injected with pCDNA-PEI were responded Fluc expression in all over the mice and it further increased to maximum Flu count on Day 4 followed reached weak count on Day 5. In addition, mice were injected with pCDNA-NCp and pCDNA-NCp-ASOR showed that Fluc expression was observed at liver along with neighbouring organs on day 4, 5 and day 6. Finally, Fluc count get reduced at liver site on Day 7, 8 and Day 9.

Saumitra Das (MCB) and N. Jayraman (OC)

Liver targeted delivery of reporter gene ‘Luciferase’ using galactose conjugated dendrimer in whole animal Hepatitis C virus primarily target liver cells. We are exploring different strategies for preferential delivery of antiviral agents into liver to study their inhibitory effect on hepatitis C virus. Asialoglycoprotein receptors (ASGPR) are abundant in hepatocytes. We are trying to use galactose ligand for liver targeting the ASGPRs. For this purpose we have studied delivery of Luciferase reporter DNA using galactose conjugated dendrimer and checked the luciferase expression by injecting the bioluminescent substrate luciferin followed by monitoring in ‘whole body imaging system’. Initial experiments showed promising results, which needs to be further optimized and preferential delivery to liver needs to be further validated using appropriate controls.

Annapoorani Rangarajan (MDG)

We are currently investigating the role of AMP-activated protein kinase AMPK in regulating epithelial-mesenchymal transition and metastasis which are linked with cancer stem cells. To study this, we have generated invasive breast cancer cell line MDA MB 231 cells expressing a GFP-expression construct or luciferase expression construct, and NBLE cells with cancer stem like-properties carrying GFP. We have begun to characterize the tumor formation and metastatic properties of these cells using the in vivo imaging facility. We are currently generating MDA MB 231 cells additionally carrying sh-AMPK constructs to study the effect of AMPK knockdown in metastasis of breast cancer cells and cancer stem cells.

Deepak Saini (MRDG)

The aim of our project is to study role of G-protein coupled receptors in cellular senescence. Studies have shown that GPCR expression and their associated signaling is altered during cellular senescence. Till date not much work has been done to evaluate communication between senescent cells and its neighboring cells per se. We will be using xenograft tumor model to study effect of senescent cells with the specific genetic background on tumorogenesis. Initially, we will establish the mouse model system to study effect of senescent cell associated signaling on tumorogenesis, wherein a mix of both senescent cells and invasive cancer cell lines will be used to study tumor formation. Both cells types tagged with fluorescent proteins will be used so that map cells by in vivo imaging to see the progress of tumorogenesis. Once the model system is established, we will use the model to study the role of the GPCR signaling pathways, by utilizing senescent cells with different genetic backgrounds like receptor knock down as well as over expressed lines to evaluate the effect of the same in the tumor progression.

Kumar Somasundaram (MCB)

We use the live animal imaging equipment to monitor the glioma tumors both intracranial and subcuteoniuos models using bioluminescence and GFP fluorescence.

Dipshika Chakravorthy (MCB) and Jagadeesh G (Aerospace Engineering)

We have developed a novel nonintrusive needleless vaccine delivery system using micro-shock waves.We have delivered fluorescent beads in BALB/C mice and biofluorescence imaging was carried out to confirm the presence of fluorescent beads in the mouse model.Fluorescent yellow-green latex beads (catalog no.L4655; Sigma-Aldrich) with a size of 1 µm were taken at a concentration of 4.6 x 109 particles per ml and delivered to the dorsal side of mice using the needle less vaccine delivery device. The excitation passband (445 to 490 nm) and emission passband (515 to 575 nm) were set for GFP. Immediately after the delivery offluorescent beads, mice were anesthetized with gaseous 5% isoflurane in O2 and imaged using a Xenogen IVIS 200 imaging system. The animals were placed in a ventral recumbent position and imaged from the dorsal aspect. The study confirms the penetration of fluorescent beads in the mouse skin.