SuppressIT® and ExpressIT® Non-Heritable Transgenics: Creating a new class of animal models using hydrodynamic injection
Introduction
Genetically modified animal models (transgenic, knock-out, knock-in and knock-down animals) represent an important tool for the study of disease processes. To obtain adult animals with heritable modifications, genetic changes are introduced into germ-line cells at the very earliest stages in development (in the egg, embryo or embryonic stem cells). Successful integration of DNA into the germ-line results in animals containing a genetic modification that can be passed to subsequent generations.
There are significant limitations to creating germ-line transgenics including embryonic, fetal or neonatal lethality, high cost, slow development times (months to years), and the high level of technical skill required and variable success rates. We have addressed these issues by developing technology to create animal models in which the genetic modification is restricted to a particular somatic tissue and/or only occurs in adult animals. siRNA and RNAi vectors can be introduced into liver or skeletal muscle cells to enable knock-down of gene expression to create SuppressIT non-heritable transgenic animal models. ExpressIT non-heritable transgenic animal models are created by hydrodynamic delivery of plasmid DNA vectors with proprietary promoters to liver or skeletal muscle to direct the long-term expression of the encoded gene, allowing for the creation of tissue-specific transgenic animals. The gene product may be localized to the expressing cells or secreted into the bloodstream.
1. SuppressIT nh-transgenic animals for gene suppression (RNA interference in vivo)
2. ExpressIT nh-transgenic animals for long-term protein expression
3. ExpressIT nh-transgenic animals expressing therapeutic genes demonstrate appropriate physiological changes
4. Genetic Immunization - nh-transgenic animals for the development of antibodies
Technical Concept
- SuppressIT nh-transgenic animals can be created by delivering siRNAs, modified siRNAs, or plasmids expressing siRNA or shRNA into liver cells in rodents. This work has been described in Lewis, et al., Nature Genetics 32 (2002) 107-108, and Wooddell, et.al., BBRC 334 (2005) 117-127.
- SuppressIT nh-transgenic animals produced using Ppara siRNAs have a genome-wide transcriptional pattern that is similar to Ppara-/- knockout mice. Both model systems displayed hypoglycemia and hypotriglyceridemia. Interestingly, fasting is required to reveal these phenotypes in Ppara-/- knockout mice but not in siRNA-treated mice. This difference may be due to abrupt knockdown in siRNA-treated mice versus absence of Ppara function throughout growth and development in Ppara-/- knockout mice and activation of compensatory mechanisms. Thus, knockdown using SupressIT has the potential to yield additional information about gene function that cannot be obtained through study of the conventional knockout mice. This work was performed in a collaborative effort with Merck & Co. scientists and has been submitted for publication.
Applications
The SuppressIT technology is particularly well-suited for creating animal models where the knock-down will interfere with embryonic or early-stage development of the animal. Models can be created in days versus months for germ-line knock-outs or knock-downs. These animal models may be useful for studying gene function, analyzing metabolic pathways and target identification. Also, SuppressIT can be used to generate knock-downs in the liver of large animals using catheter based hydrodynamic delivery where germ-line transgenics would be cost prohibitive. It may also be valuable to compare the difference between animals where gene suppression occurs during development as in knockout mice versus only in adulthood.
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Technical Concept
- ExpressIT nh-transgenic animals have been created using the proprietary pLIVE™ vector with high level expression of transgene-encoded proteins in the bloodstream (examples include: erythropoietin, apolipoprotein E, secreted alkaline phosphatase, factor VIII, factor IX, a1-antitrypsin, a-galactosidase, insulin, leptin, CNTF, TNF-a, IL10, IL12, IFN-b, IFN-g). Animal models can be developed with transgene-encoded proteins specifically expressed in liver hepatocytes or limb skeletal muscle cells (myofibers). Expressed proteins may be localized or secreted.
Applications
ExpressIT technology is particularly well-suited for models where expression of the target gene would interfere with embryonic or early-stage development of the animal. It is also a cost-effective approach for use in larger animals where germ-line transgenics are cost-prohibitive. Animal models can be created using ExpressIT technology in days or weeks versus months for germ-line transgenics. Applications include both expression of the protein within the target cells (either hepatocytes or skeletal muscle) or secretion to study systemic effects. In addition, SuppressIT techniques can be used to knock-down expression of the target gene as an experimental control.
ExpressIT animal models provide a rapid and cost effective approach to studying the physiological impact of localized or systemic expression of a target gene. These animal models may also be useful for target identification.
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Technical Concepts
- ExpressIT nh-transgenic animals can be used with knock-out animals to “rescue” the genetic knockouts, e.g. ApoE expression in ApoE knockout mice. This provides an important additional tool or control for using these animal models in target discovery.
- ExpressIT nh-transgenic animals expressing a gene demonstrate appropriate long term phenotypes, e.g. expression of erythropoietin increases hematocrit.
Applications
In most research applications, the goal is to relate gene expression to phenotype. Animal models generated using ExpressIT technology exhibit appropriate phenotypes. However, the phenotype may be different when the gene expression is induced in an adult animal using ExpressIT techniques versus having the gene expression present through embryonic and early development. The ExpressIT animal models may be more representative of the phenotypes seen in disease that only develops in adulthood.
ExpressIT animal models may be an attractive alternative or complement to germ-line transgenics for target identification where the endpoint is a phenotypic change.
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Technical Concept
- nh-transgenic animals were created by hydrodynamic injection of pDNA vectors encoding immunogenic proteins. Strong antibody responses to the expressed foreign proteins were detected and quantified. Benefits include a reduced process time to generate antibodies and eliminating the need to purify proteins or synthesize peptides for injection. Also intrinsic to the process, specific antibodies are generated that recognize proteins with various post-translational modifications (i.e. different phosphorylated and/or glycosylated forms). See our publication, Bates, et.al., BioTechiques 40:2 (Feb. 2006) 199-207, and more detailed discussion (more information).
Applications
Genetic immunization provides unique benefits for developing antibodies against antigens where the immunogen is difficult to produce, administer or where antibodies against post-translational modifications are desired. It is especially useful in generating antibodies to post-translationally modified proteins where the optimal immunogenic site has not been identified.
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Summary
It is now possible to generate SuppressIT or ExpressIT non-heritable transgenic animals quickly and cost-effectively as either an alternative or complement to germ-line transgenics. The two proprietary scientific breakthroughs that enable this new capability are the hydrodynamic delivery methodology and the development of the pLIVE plasmid DNA vector capable of long term, sustained transgene expression in hepatocytes. Combining these technologies enable generation of adult nh-transgenic animals with directed knock-down of target genes or expression of transgenes in liver or muscle for weeks or months following a single hydrodynamic delivery. In addition, it is possible to express proteins that are efficiently secreted into the bloodstream. Novel adult animal models that might be created using this technology include: disease models resulting from over-expression of mutated endogenous gene products (dominant negative proteins, modified receptors), disease models resulting from expression of single chain antibodies (against an endogenous antigen), ‘genetic rescue’ of knock-out mice or rats, and large animal nh-transgenic models. These nh-transgenic models provide a useful tool for studying gene expression, analyzing metabolic pathways and for target identification.
This technology can also be employed for genetic immunization, including generating monoclonal antibodies to post-translational protein modifications. Also, by using pDNA vectors that encode and express weakly immunogenic proteins (normal polymorphisms) it will be possible to study many of the intricacies of the native immune response in adult animal models.
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