In Vivo Transfection

Delivery to Animal Models

In vivo transfection is the ability to deliver a payload to specific cells within a living organism, maintain the ability to release the cargo upon entry into the cell and provide degradaton protection to the payload.  Delivery of a payload in a living animal brings forth extreme challenges compared to cultured cells.  When conducting an experiment with living cells, there are two categories to classify all experiments. An experiment is either termed “in vitro” or “in vivo“. See the diagram below for an easy comparison:

In vitro is defined as “within glass”; In vivo is defined as “within the living”. If you’d like a more in-depth explanation of the differences between in vitro and in vivo, visit

In vitro refers to experiments setup in a flask or dish.  The living portion of an in vitro experiment is typically an immortalized cancer cell line.  In vivo experiments are conducted on living multicellular organisms.  Both types of experiments focus on using living cells as the platform to study drug candidate efficacy.


Products for in vivo transfection are commercially offered by multiple private companies. An American based company that provides in vivo products and services is Altogen Labs.

In vivo studies always brings forth unforeseen complications not typically encountered during in vitro experimentation. To aid researchers with in vivo delivery of their test article, commercially available reagents help to side-step some of the common pitfalls.  These include items such as tissue clearance, specificity, stability of the DNA/RNA mimic, efficiency, half-life, immune response and toxicity.  Typically, delivery formulations can be altered to include some of these molecules:

    • • Lipid-based
    • • Polymer-based
    • • PEGylated
    • • Nanoparticles


Cell line-derived xenograft animal models (CDX mouse models) enable researchers to determine in-life bioavailability and efficacy of novel drug compounds in a specific cancer type.  Subcutaneous and orthotopic engraftment of cancer cell lines in immunocompromised mice provide a valuable platform that mimics course of action in human cancer treatment.  These initial steps in the life of a cancer therapeutic are the typical route of assessment for future, clinically approved anti-cancer drugs.  However, with any development program, careful considerations regarding the following topics must be addressed in order to circumvent pitfalls.


Transfection reagents typically rely on positively charged molecules in order to achieve successful gene transfer activity in vitro.  Although acceptable for in vitro applications, a positively charged structure brings forth problems when systemically injected and therefore cannot be utilized as an in vivo delivery agent.  Positively charged complexes are quickly cleared from circulation upon injection and the lack of the complex in circulation within the animal means the inability of the active pharmaceutical ingredient (API) to reach the target tissue.

By incorporating modifications to the exterior of the transfection reagent (i.e. delivery agent) results in added protection from systemic clearance, highly modified reagent formations can often lead to a lack of target effect due to the inability of the reagent to release the payload endosomally.  Although protection from degradation and clearance, lack of triggering an immune response and injection route are all important points to consider, the ability to release the delivered cargo into the cytoplasm of the targeted cells is the most important aspect to consider in choosing an in vivo transfection reagent.


Xenograft studies require consideration or multiple factors, including the selection of the appropriate animal model and the best drug administration route of the final drug formulation.  Here are some common routes of delivery:

    • • Intravenous
    • • Intratracheal
    • • Continuous infusion
    • • Intraperitoneal
    • • Intratumoral
    • • Oral gavage
    • • Topical
    • • Intramuscular
    • • Subcutaneous
    • • Intranasal
    • • Micro-injection techniques
    • • Pump-controlled IV injection

Additionally, the dosing frequency, duration and engraftment site play a crucial role in drug efficacy.


Here are typical xenograft study design and data collection options:

    • • Tumor Growth Delay
    • • Tumor Immunohistochemistry
    • • Quantitation of Target Genes (i.e. histology, mRNA and protein expression levels)
    • • Blood Chemistry Analysis
    • • Tumor Growth Inhibition
    • • Toxicity and Animal Survival
    • • Imaging Studies: fluorescence-based whole body imaging and tumor MRI