We are working with a endoplasmatic reticulum resident protein, and we have made a GFP fusion of this protein.
We want to confirm the ER lumen localization .
I was looking at the wormbase and I saw the C.elegans strain WH327, which has the trangene ojls23.
Regarding that, I have a doubt with nomenclature;
Are the following nomenclature equivalent:
[pie-1::gfp-C34B2.10] and [SP12::GFP]
Another question is what is the meaning of the hyphen between gfp and C34B2.10?
I will very much appreciate if anyone can help me with these doubts
Thank you very much in advance
No, they are not equivalent.
The first construct, pie-1::gfp-C34B2.10 is likely to be an N-terminal GFP tag. The second construct, SP12::GFP is a C-terminal GFP tag.
The genotype of the strain WH0327 is: WH0327: unc-119(ed3); ojIs23[SP12::GFP unc-119(+)]
This is how you “read” the genotype:
The transgene ojIS23 is introduced into the mutant unc-119(ed3). The complete description of the exact composition of ojIS23 is given in parenthesis. The transgene ojIS23 is made up of two types of plasmids:
(1) SP12 fused with GFP (Green) (2) wildtype copy of unc-119 gene [denoted as unc-119(+)]. The first plasmid, SP12 fused with GFP is the one that you are interested in - it lights up ER. The second plasmid is a selection marker.
In ‘ojIS23’, the first two letters ‘oj’ denotes the lab in which this transgenic animal was generated. In this case its from Dr. John White’s lab. The next two letters, ‘IS’ denotes the nature of transgene and means the transgene is ‘integrated into the chromosomal DNA’. The transgene can exist in one of the 3 ways: either the transgene can ‘float’ freely and behave like a small chromosome (in which case the transgene is called ‘extrachromosomal array’ and is represented as ‘Ex’) or the transgene can get stably integrated into the chromosomal DNA (in which case it is denoted as ‘IS’). Or a Single copy of a target transgene is integrated into a specific location of a genome (Mos insertion)
The genotype pie-1::gfp-C34B2.10 indicates that pie-1 promoter drives the expression of the GFP fused to the N-terminal side of C34B2.10.
Guna gave a nice summary. Let me add that C34B2.10 is SP12, so both should light up the ER. As Snug indicated the gfp-C34B2.10 is likely an N-terminal fusion. The correct nomenclature would use double colons rather than the dash to indicate fusion, but sometimes that isn’t strictly followed.
If you need to express the protein in a specific cell or at a specific time at which the pie-1 promoter is not active, you could request the DNA from a lab that has used it (such as the Starr lab), or perhaps they have a strain that isn’t at the CGC but would be useful to you.
One last small point, the strain was made by microparticle bombardment with a single plasmid that has both the unc-119 rescue and expression construct. Given that both proteins are GFP fusions, you may want to do antibody staining.
I also have lots of questions about nomenclature. Is there a link where you could find the rules that are commonly used? I find it a bit confusing.
Is there a link where you could find the rules that are commonly used?
The first formal nomenclature rules were published [url=http://www.ncbi.nlm.nih.gov/pubmed/292825]in a 1979 paper[/url] and were compiled in the back of the CSHL Press books "The Nematode [i]Caenorhabditis elegans[/i]" (1988) and "[i]C. elegans[/i] II" (1997), commonly called "Worm I" and "Worm II", respectively. [url=http://www.ncbi.nlm.nih.gov/books/NBK20203/]The version in the back of Worm II is available online[/url].
The rules have been expanded and updated since those publications (although I’d suggest it’s easier to learn the nomenclature by starting with the simpler version to be found in the back of Worm II, which will cover most day-to-day issues), and this expanded and updated version can be found at WormBase.
You should probably be aware that there is significant person-to-person variation in how transgene constructs are named and described. The allele name of a given transgene is decided according to strict rules, and these rules dictate that its contents are to be listed (if at all) within a pair of square brackets, but the contents of that transgene may not be readily, consistently, and correctly discerned from what is put within the square brackets.
Thanks a lot for these links, it’s really helpful!