Gfp-cdna
Encyclopedia
The GFP-cDNA project documents the localisation of protein
s to subcellular compartments of the eukaryotic cell
applying fluorescence microscopy. Experimental data are complemented with bioinformatic
analyses and published online in a database
. A search function allows the finding of proteins containing features or motifs of particular interest. The project is a collaboration of the research groups of Rainer Pepperkok at the European Molecular Biology Laboratory
(EMBL) and Stefan Wiemann at the German Cancer Research Centre (DKFZ).
(ORF) are tagged with Green Fluorescent Protein
(GFP) and expressed in eukaryotic cells. Subsequently, the subcellular localisation of the fusion protein
s is recorded by fluorescence microscopy.
Steps:
technologies which are free of restriction enzyme
s. In this respect those that utilise recombination
cloning (Gateway of Invitrogen or Creator of BD Biosciences) have proved to be the most suitable. This cloning technology is based on recombination mechanisms used by phages to integrate their DNA
into the host genome
. It allows the ORFs to be rapidly and conveniently shuttled between functionally useful vector
s without the need for conventional restriction cloning. In the cDNA-GFP project the ORFs are transferred into CFP/YFP
expression
vectors. For the localisation analysis both N- and C-terminal fusions are generated. This maximises the possibility of correctly ascertaining the localisation, since the presence of GFP may mask targeting signal
s that may be present at one end of the native protein.
s). Particularly interesting ORFs are also screened for localisation in PC12 cells and hippocampal
neuron
s.
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s to subcellular compartments of the eukaryotic cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
applying fluorescence microscopy. Experimental data are complemented with bioinformatic
Bioinformatics
Bioinformatics is the application of computer science and information technology to the field of biology and medicine. Bioinformatics deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, software...
analyses and published online in a database
Database
A database is an organized collection of data for one or more purposes, usually in digital form. The data are typically organized to model relevant aspects of reality , in a way that supports processes requiring this information...
. A search function allows the finding of proteins containing features or motifs of particular interest. The project is a collaboration of the research groups of Rainer Pepperkok at the European Molecular Biology Laboratory
European Molecular Biology Laboratory
The European Molecular Biology Laboratory is a molecular biology research institution supported by 20 European countries and Australia as associate member state. EMBL was created in 1974 and is an intergovernmental organisation funded by public research money from its member states...
(EMBL) and Stefan Wiemann at the German Cancer Research Centre (DKFZ).
What kinds of experiments are made?
The cDNAs of novel identified Open Reading FramesOpen reading frame
In molecular genetics, an open reading frame is a DNA sequence that does not contain a stop codon in a given reading frame.Normally, inserts which interrupt the reading frame of a subsequent region after the start codon cause frameshift mutation of the sequence and dislocate the sequences for stop...
(ORF) are tagged with Green Fluorescent Protein
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP) and expressed in eukaryotic cells. Subsequently, the subcellular localisation of the fusion protein
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
s is recorded by fluorescence microscopy.
Steps:
1. Large-scale cloning
Any large-scale manipulation of ORFs requires cloningCloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
technologies which are free of restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s. In this respect those that utilise recombination
Genetic recombination
Genetic recombination is a process by which a molecule of nucleic acid is broken and then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in non-homologous end joining. Recombination is a common method...
cloning (Gateway of Invitrogen or Creator of BD Biosciences) have proved to be the most suitable. This cloning technology is based on recombination mechanisms used by phages to integrate their DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
into the host genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. It allows the ORFs to be rapidly and conveniently shuttled between functionally useful vector
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
s without the need for conventional restriction cloning. In the cDNA-GFP project the ORFs are transferred into CFP/YFP
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
vectors. For the localisation analysis both N- and C-terminal fusions are generated. This maximises the possibility of correctly ascertaining the localisation, since the presence of GFP may mask targeting signal
Signal sequence
Signal sequence can refer to:*Protein targeting*Signal peptide*DNA uptake signal sequence...
s that may be present at one end of the native protein.
N-Terminal Fluorescent Fusions
Insert your gene of interest into the MCS upstream of the fluorescent protein gene, and express your gene as a fusion to the N-terminus of the fluorescent protein.C-Terminal Fluorescent Fusions
Insert your gene of interest into the MCS downstream of the fluorescent protein gene, and express your gene as a fusion to the C-terminus of the fluorescent protein.2. Transfection of eukaryotic cells, Expression
The fusion vectors are transfected in Vero cells (monkey kidney fibroblastFibroblast
A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for animal tissues, and plays a critical role in wound healing...
s). Particularly interesting ORFs are also screened for localisation in PC12 cells and hippocampal
Hippocampus
The hippocampus is a major component of the brains of humans and other vertebrates. It belongs to the limbic system and plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. Humans and other mammals have two hippocampi, one in...
neuron
Neuron
A neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
s.
3. Protein localisation
At different time points, the subcellular localisation of the fusion proteins is recorded via fluorescence microscopy. At the end of the live cell imaging, the cells can still be fixed and colocalisation experiments made.4. Bioinformatic Analysis
As the sequence of the cDNAs is known, bioinformatics can make predictions regarding the localisation and function of the encoded protein. The bioinformatics analysis is facilitated by the bioinformatic search engine Harvester.5. Assignment of subcellular localization category
Results from the N- and C-terminal fusions are assessed and in turn these data are compared to the bioinformatic predictions. A final subcellular localisation (from approximately 20 categories) is then assigned for each ORF. Similar localisations with both N- and C-terminal constructs provide a higher degree of reliability of the result. For those ORFs where the two fusions do not give a similar localisation pattern, a series of other criteria, including bioinformatic predictions, are considered. Occasionally a clear cut localization cannot be assigned.