1. Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer. Liposomes can be coated with Polyethylene Glycol or PEG which screens the liposome from the immune cells. www.eurofedlipid.org Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Leading Developer and Manufacturer of In Vivo and DNA Transfection Kits, Transfection Reagents and Electroporation Delivery Products Products > PEG-Liposome In Vivo Transfection Kit

2. Altogen Biosystems is developing optimized transfection technologies for a specific cell line by applying expertise in combinatorial chemistry, molecular biology, and cell biology. The successful use of liposome compounds to deliver exogenous nucleic acids to specific biological system is dependent upon several factors including lipid formulation, charge ratio, particle size and the method of liposome preparation. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Liposome Transfection Products > PEG-Liposome In Vivo Transfection Kit

3. www.slideshare.net Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Mechanism of Action

4. Advances in liposome research have produced the biotechnology to allow liposomes to avoid detection by the body's immune system, specifically, the cells of reticuloendothelial system (RES). These liposomes are constructed with PEG (Polyethylene Glycol) studding the outside of the membrane. The PEG coating is inert in the body and allows for longer circulatory life for the drug delivery mechanism. In addition to a PEG coating, these types of liposomes also have some sort of biological species attached as a ligand to the liposome, to enable binding via a specific expression on the targeted drug delivery site. These targeting ligands could be monoclonal antibodies (making an immunoliposome), vitamins, or specific antigens. Targeted liposomes can target nearly any cell type in the body and deliver drugs that would naturally be systemically delivered. Naturally toxic drugs can be much less toxic if delivered only to diseased tissues. www.wikipedia.org Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Development of Therapeutic Agents

5. PEGylated liposome based reagent developed and manufactured by Altogen Biosystems No detectable toxicity or inflammatory response due to biodegradable PEG modification Complexes are stable in serum for at least 16 hours Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Product Details

6. Efficient delivery to the spleen, kidney, pancreas, liver, and multiple tumor types via systemic administration Efficient delivery of negatively charged molecules including siRNA (shRNA, microRNA), plasmid DNA, and protein Functionally tested in mice Applicable for plasmid DNA/siRNA(shRNA) co-injection Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Product Details

7. Modes of Administration Systemic intravenous (i.v.) injection Direct intratumoral (i.t.) injection Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Product Details

8. Figure 1. Systemic administration (i.v.) of Liposome-PEG based In Vivo reagent conjugated with siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues were collected and RNA isolated 48 hours after first injection. Samples were analyzed by qRT-PCR for Lamin A/C gene expression levels. Ribosomal RNA levels were used to normalize the Lamin A/C data. Data are means ± SD (n=6). Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data

9. Figure 2. Intratumoral administration (i.t.) of Liposome-PEG based In Vivo reagent conjugated with siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues were collected and RNA isolated 48 hours after first injection. Samples were analyzed by qRT-PCR for Lamin A/C gene expression levels. Ribosomal RNA levels were used to normalize the Lamin A/C data. Data are means ± SD (n=6). Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data

10. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data Figure 3. Systemic administration (i.v.) of PEG-Liposome In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues (Liver, Brain, Muscle, Kidney, Tumor, Heart, Spleen, Lung) were collected and total protein fraction isolated 72 hours after first injection. Samples were analyzed by Western Blot Analysis for Lamin A/C gene expression levels.

11. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data Figure 4. Systemic administration (i.v.) of PEG-Liposome In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues (Liver, Brain, Muscle, Kidney, Tumor, Heart, Spleen, Lung) were collected and total protein fraction isolated 72 hours after first injection. Samples were analyzed by Western Blot Analysis for Lamin A/C gene expression levels.

12. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data Figure 5. Systemic administration (i.v.) of PEG-Liposome In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues (Liver, Brain, Muscle, Kidney, Tumor, Heart, Spleen, Lung) were collected and total protein fraction isolated 72 hours after first injection. Samples were analyzed by Western Blot Analysis for Lamin A/C gene expression levels.

13. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Data Figure 6. Systemic administration (i.v.) of PEG-Liposome In Vivo Transfection Reagent conjugated with 80 ug of chemically modified siRNA targeting Lamin A/C mRNA or non-silencing control siRNA following the recommended protocol. Tissues (Liver, Brain, Muscle, Kidney, Tumor, Heart, Spleen, Lung) were collected and total protein fraction isolated 72 hours after first injection. Samples were analyzed by Western Blot Analysis for Lamin A/C gene expression levels.

14. Contents and Shipping: PEG-Liposome based in vivo transfection reagent is supplied in liquid form at a concentration of 2.8 mg/ml. Transfection Enhancer is supplied in liquid form at a concentration of 1 mg/ml. Shipped at ambient temperature. Description: PEGylated (polyethylene glycol 2000) cationic lipid liposome-based In Vivo Transfection Reagent is a proprietary formulation of lipid-functionalized PEG complex optimized for in vivo delivery of siRNA, plasmid DNA, and proteins. Product Qualification: Reagents are tested for functional activity, absence of nuclease contamination and microbial contamination. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Product Details

15. Reagent Properties: PEG-Liposome conjugated complexes are stable in serum for at least 16h. No detectable toxicity or inflammatory response due to biodegradable PEG modification. Efficient delivery to the spleen, kidney, pancreas, liver, and multiple tumor types via systemic administration. Intended Use: For research use only. Not intended for any diagnostic or therapeutic use. General Guidelines: Handle animals and conduct experiments according to national regulations and approvals by the local IACUC and ethics committee. Please note that mouse tail vein injection may require skills, experience, and associated equipment (e.g. restraining device). All persons handling animals should be properly trained. A miRNA molecule is single-stranded and 22 nucleotides long; it binds to a complementary segment of RNA to create a double-stranded molecule. Both of these molecules may occur naturally in the cell as well as being artificially introduced, and their effects may last three to seven days. Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Protocol Details Continued

16. PEGylated liposome based reagent developed and manufactured by Altogen Biosystems No detectable toxicity or inflammatory response due to biodegradable PEG modification Complexes are stable in serum for at least 16 hours Efficient delivery to the spleen, kidney, pancreas, liver, and multiple tumor types via systemic administration Efficient delivery of negatively charged molecules including siRNA (shRNA, microRNA), plasmid DNA, and protein Functionally tested in mice Applicable for plasmid DNA/siRNA(shRNA) co-injection Altogen Biosystems  848 Rainbow Blvd #823  Las Vegas  NV  89107  USA Telephone  702 349 6103  Fax  702 989-0841  email  techserv@altogen.com Products > PEG-Liposome In Vivo Transfection Kit Summary