Cloning a gene without the sequence of the target species - (Jun/08/2005 )
I need to clone a gene and overexpress into rat cells to see the effects of the gene. However, in the pubmed, only mouse and human sequences are available and for the rats, only predicted sequence is available. Then, what should I do? Use the sequence of human or mouse to design the primers and use the rat DNA as the template? or use the predicted sequence of rats to design primers? Are there any follow-ups that I have to do? Thanks
Sorry.... I not really understand your problem.
First, what gene you want to clone into rat, is human gene or mouse gene?
Please tell me your project more detail so that I can help you : )
Thanks for your reply.
I have to clone a gene that should be present in rats, but the rat sequence of the gene is not available on the pubmed database and only the PREDICTED rat sequence, human sequence and mouse sequence are available.
However, my animal model is rat and I will use rat DNA as the template. My target gene will be overexpressed in rat cell lines. What i am concerned is how to clone the gene and the problems behind.
If I use the predicted rat sequence, it may be challenged for not having any meanings, since I even dont know if this is really my target gene. My colleague suggests me to make a clone by the mouse sequence to see if the trend of the two species are similar and also their protein alignment.
For using the mouse or human sequence to design the primers for clioning, even if the gene can be cloned, it may not be the same with the one in rats. Then, I may have to do RACE,, right?
Thats what I am concerned. And what should I do to clone the gene? Correct me if I am wrong.
Your friend made a good suggestion. The rat genome isn't nearly as complete/well annotated as either the human or mouse genomes in NCBI. In order to verify if the predicted gene location is likely to be correct, you can do sequence alignments between human/mouse and rat of both the DNA/RNA and protein sequences for your gene of interest. Using either Mapviewer or the UCSC genome browser will give you the complete DNA genomic sequence. You can also compare the species directly in NCBI using the Taxonomy function.
I understand your pain when working with the rat genome Good luck and post back with your results.