GFP Lentivirus
BioInnovatise Viral Vector Team
Updated September 16, 2024
GFP (Green Fluorescent Protein) is one of the most common open reading frames (ORFs), reporters, and tags in molecular biology, and specifically lentivirus packaging, for many reasons. Our scientists have decided to answer some common lentivirus GFP related questions.
Note: Although there are Green Fluorescent Protein ORFs, reporters, tags, inducible reporters and inducible systems, the phrase “GFP lentivirus” refers most often to a reporter.
Why is GFP lentivirus so frequently used in molecular biology and lentivirus packaging? The answer has to do with GFP’s efficient tracking of viral transduction, allowing researchers to identify successfully transduced cells by their fluorescence, simplifying downstream analysis. Here is a breakdown of the many advantages to using a GFP reporter in lentivirus constructs or packaging:
Non-invasive and Visual Detection
- GFP allows live-cell imaging because it emits bright green fluorescence when exposed to blue or UV light. This makes it easy to monitor and track cellular processes in real time without harming the cells.
Intrinsic Fluorescence
- Unlike other reporters, GFP doesn’t require additional cofactors, substrates, or external dyes to fluoresce. The fluorescence is an intrinsic property of the protein, simplifying experimental setups.
Versatile and Universal
- GFP can be fused to many proteins of interest to study protein localization, expression, and interactions in a wide range of organisms (bacteria, yeast, plants, animals).
- It works in various biological systems and is easily incorporated into vectors, such as lentiviruses, to label cells or proteins.
Mutants and Variants
- GFP has been optimized and modified to create various mutants with different fluorescence intensities and colors (like YFP, CFP, etc.), providing a wide range of applications for multiplexing and dual-reporter systems. See below variations and derivations of GFP.
Low Toxicity
- GFP is generally non-toxic to cells, making it suitable for long-term studies in live cells or organisms. It does not interfere significantly with normal cellular processes.
Quantitative Reporting
- GFP can be used as a quantitative marker, allowing researchers to measure gene expression levels or protein abundance by tracking fluorescence intensity.
Historical Legacy and Popularity
- Since its discovery and its widespread use in Nobel-winning research, GFP has become a well-characterized, trusted tool in the molecular biology community.
- As a result, it’s extensively used, and protocols for GFP are well-established.
GFP Reporters, Tags, Open Reading Frames, Inducible Reporters, and Inducible Systems
As we mentioned above, GFP lentivirus frequently refers to a reporter system, however there are other green fluorescent proteins that can be added to a plasmid DNA construct during the molecular cloning process.
1. Reporter System (Most Common Use)
- GFP lentivirus is frequently used as a reporter to track cells that have been successfully transduced by the lentivirus during lentivirus transfection. Researchers use GFP to monitor the efficiency of viral delivery, transgene expression, or to visualize infected cells in real time.
- When GFP is used as a reporter, it is usually expressed under the control of a constitutive promoter (like CMV or EF1α) for stable and continuous fluorescence.
2. Tag
- In this context, the GFP-lentivirus delivers a gene encoding a fusion protein (protein of interest + GFP).
- This allows researchers to study the localization, trafficking, or interactions of the target protein within living cells. GFP-tagged proteins are often used for protein tracking and visualization.
3. ORF
- GFP can be delivered as an ORF via lentivirus, but when people talk about “GFP lentivirus,” they typically emphasize its role as a reporter or tag rather than just an ORF.
- In cases where GFP is delivered as an ORF, it is often in the context of overexpressing GFP itself or creating a tool for screening.
4. Inducible Reporter/System
- In some cases, GFP can be part of an inducible system, where its expression is controlled by an inducible promoter such as a tetracycline-responsive promoter.
- In this system, GFP expression can be turned on or off in response to specific inducers, allowing temporal control over gene expression and tracking. However, this is less common unless researchers specifically need inducibility.
GFP Lentivirus Derivations and Alternatives
Because GFP has proven to be such a versatile protein for a vast amount of research purposes, there have been several variations of the original protein produced and engineered to achieve similar yet distinctive end results. Below are some of the GFP variations that are commonly used in research.
Reporter: eGFP (Enhanced Green Fluorescent Protein)
- Size: ~720 bp
- Description: A modified version of the original GFP from Aequorea victoria jellyfish. Enhanced for brighter fluorescence and faster folding.
- Origin: Aequorea victoria jellyfish, with mutations to improve brightness and reduce issues with folding in mammalian cells.
Reporter. EYFP (Enhanced Yellow Fluorescent Protein)
- Size: ~717 bp
- Description: A variant of GFP optimized for yellow emission (~527 nm), with improved brightness and photostability.
- Origin: Derived from Aequorea victoria, with mutations for yellow fluorescence.
Reporter. CFP (Cyan Fluorescent Protein)
- Size: ~720 bp
- Description: A GFP variant optimized for cyan emission (~476 nm), used in FRET (Förster resonance energy transfer) experiments.
- Origin: Engineered from Aequorea victoria GFP.
Reporter. Venus
- Size: ~720 bp
- Description: A variant of yellow fluorescent protein (YFP) with faster maturation and less sensitivity to pH.
- Origin: Derived from GFP.
Reporter: BFP (Blue Fluorescent Protein)
- Size: ~720 bp
- Description: A blue variant of GFP with an emission peak around 447 nm. Used in FRET applications and for multi-color labeling.
- Origin: Engineered from GFP.
ORF: EGFP (Enhanced Green Fluorescent Protein)
- Size: ~720 bp
- Description: An enhanced variant of GFP with improved brightness and stability.
- Origin: Modified version of Aequorea victoria GFP.
ORF: BFP (Blue Fluorescent Protein)
- Size: ~720 bp
- Description: Fluorescent protein that emits blue light, often used in multi-color assays.
- Origin: Engineered from GFP (Aequorea victoria).
ORF. mVenus
- Size: ~717 bp
- Description: A variant of YFP (Yellow Fluorescent Protein) with increased brightness and faster maturation.
- Origin: Engineered from Aequorea victoria GFP.
ORF. Cerulean
- Size: ~720 bp
- Description: A bright cyan fluorescent protein used in FRET and multi-color fluorescence studies.
- Origin: Derived from GFP (Aequorea victoria).
ORF. eCFP (Enhanced Cyan Fluorescent Protein)
- Size: ~717 bp
- Description: A variant of cyan fluorescent protein (CFP) used in multi-color assays.
- Origin: Derived from GFP (Aequorea victoria).
ORF. BFP2
- Size: ~717 bp
- Description: Improved version of BFP with enhanced brightness and stability.
- Origin: Engineered from GFP (Aequorea victoria).
Tag. GFP-Tag
- Size: ~720 bp
- Description: A GFP fusion tag for visualization of protein localization and real-time expression in cells.
- Origin: Aequorea victoria.
Inducible Reporter. pBAD-GFP
- Size: ~700 bp for GFP, ~1.2 kb for pBAD regulatory elements
- Description: A fusion of the arabinose-inducible pBAD promoter with the GFP reporter gene, allowing visual tracking of expression in response to arabinose.
- Origin: Synthetic construct derived from the E. coli arabinose operon.
Inducible Reporter. GFP (Green Fluorescent Protein) Reporter
- Size: ~720 kb
- Description: GFP produces green fluorescence when excited by UV or blue light, making it a versatile reporter in various inducible systems.
- Origin: Jellyfish (Aequorea victoria).
Inducible Reporter. GFP (Green Fluorescent Protein) Reporter
- Size: ~720 kb
- Description: GFP produces green fluorescence when excited by UV or blue light, making it a versatile reporter in various inducible systems.
- Origin: Jellyfish (Aequorea victoria).
GFP Usage in AAV, Adenoviral, and Retroviral Vectors
Can GFP be used in other viral vectors such as adenovirus, adeno-associated virus, and retrovirus? Sure! However for a variety of reasons, GFP works best in lentiviral applications. Here are some of the limitations of GFP in other viral vectors:
Adenoviral Vectors
- Adenoviral vectors provide high transduction efficiency and strong expression of GFP, but they do not integrate into the host genome. This leads to transient expression, which may be ideal for short-term studies where temporary expression of GFP is needed.
- Adenoviruses can provoke strong immune responses, especially in vivo, which may limit their use in certain applications or necessitate immune suppression.
AAV Vectors
- AAV vectors can provide stable GFP expression for extended periods, but they typically remain episomal (i.e., they do not integrate into the host genome).
- AAV vectors are less immunogenic than adenoviruses, making them safer for in vivo applications, including gene therapy.
- AAV vectors have a relatively small packaging capacity (~4.7 kb), which can limit their use when multiple genes or larger inserts are needed.
Retroviral Vectors
- Like lentiviruses, retroviruses integrate into the host genome, but they are restricted to transducing dividing cells. This makes them less versatile than lentiviruses, especially when transduction of non-dividing cells is required.
- Retroviruses may not transduce certain cell types as efficiently as lentiviruses.
Can I Add GFP to My Lentiviral Construct And Downstream Packaged Particles?
Of course! Our molecular cloning team is more than able to add a GFP reporter to your plasmid DNA construct to help with your downstream applications and visual analysis. If you are interested in packaging your GFP lentivirus construct in a lentiviral particle, learn more about our lentivirus packaging services.
What if I already have a different reporter or tag? No worries, our molecular cloning team can add an extra reporter or tag to any plasmid DNA construct up to 18 kbp.
GFP Lentivirus Control Virus Productions
If you are looking for control viruses to use in your research that contain a GFP, or GFP variation, our team can produce control viruses. Just let us know what promoter, optional tags, and generation of lentivirus you are interested in when starting a production.
Want to learn more about the latest in lentivirus based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on lentivirus-based research.
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