I am having a mouse generated that is supposed to over-express a peptide in a cardiac-restricted manner, using a cardiac-specific alpha MHC promoter. The construct contains the promoter and downstream the N-terminal HA-tagged peptide sequence. I have 30 founders and have done standard genotyping with primers specific to the promoter (fwd) and a portion of the tag and peptide sequence (reverse). I have five hits, but need to do copy number analysis as well and will be doing this analysis on the F1 generations. I'm not sure what I should be using as a calibrator. Some methods use southern blotting, while others use qpcr, but in a lot of cases, some calibrator with known transgene copies is used, but I don't have anything that I can think of to use as a calibrator. Any insights on how this is usually done would be greatly appreciated.
I would suggest to first look on gene expression pattern in F1 rather than to spend time on determining the copy number. I guess that when a mouse will not express the transgene where you expect it to be expressed, it's useless for you.
I would start with IHC, and if you are lucky enough to see tissue-specific expression, than determine the protein levels using western blot.
@ Lech,
My working model organism is plants. I am interested to know (in animal field):
If you have transgenic mice with multiple transgene insertions, and it affects transgene expression (gene silencing), do people choose to mate these transgenic mice to 'wild (non-transgenic)' mice, and get progeny with 'single-copy' transgene (by genetic segregation) for evaluating their expression level again? Or you just toss these transgenic mice away?
In plants, we do so (backcrossing to wild-type) sometimes.
Dear Yuan,
To answer your question: yes, founder mice (obtained by zygote injection of transgene DNA) genotyped positively for the transgene are mated to wild-type mice, and the progeny (F1) are analyzed first (typically you need to sacrifice a mouse to analyze it). By analyzing the F1 you also decrease the chances that your gene is in multiple places in the genome and make sure it does not impair reproduction.
When a transgenic animal (most commonly mouse) is generated by random integration of the transgene, the transgene is integrated in multiple (tandem) copies in a single locus. The randomly integrated transgenes are almost always in multiple tandem copies, in one genomic locus, inherited as one allele. The likelihood that you get such tandem integrations in more than one locus is very low (though it happens sometimes, and in such case you may treat the F0 as a "double founder", giving rise to two independent lines.
How do you breed transgenic plants (technically)? Do you transfer the pollen from WT to transgenic flowers, and then collect the seeds? Forgive me my ignorance.
Dear Ryan, we use qPCR for evaluating the copy number of transgenic integrates. Look at the following publication: http://www.ncbi.nlm.nih.gov/pubmed/22140568 - there we used ApoB as reference gene to normalize the DNA amount. You should also clone the PCR fragment and use a serial dilution of this plasmid to calculate the absolute copy number (I would recommend to ad similar amounts of wt DNA in each of the dilutions). Beside that I agree with Lech that I would first look at some individuals for the exprected expression pattern).
regards - kai
we used the same method as described above by Kai but in the mouse (different gene of intereset and also diff. gene for calibration but our modell is the mouse so you might use the same primers for internal controll) you can check the description of the method in this publication: http://link.springer.com/article/10.1007%2Fs11248-007-9108-9
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