Copy number of a transgene
Tips for using Southern blot to determine the copy number
- choose a restriction enzyme cut inside the transgene
- the probe should be at the one end of the transgene but not cross the restriction site.
The following is an example (by courtesy of Dr. Ernie Canalis)
If it is a single copy integration, Southern will shows a single band with an unknown length which is determined by next RV site in the mouse genome.
If it is a multiple copy integration with tandem arrangement, Southern will shows a 3.3 kb band and a band with an unknown length.
The bands for multiple copy integration with head to head or tail to tail arrangement can also be predicted by the same way.
3.3 kb
Southern blot of a founder (F) and his progenies (1-16).
There are three integration events:
Integration A: #7 shows a single band of 4.5 kb indicating a single copy transgene integration.
Integration B: #4 shows multiple copies of integration with 3.3 kb and 5.6 kb bands.
Integration C: #1, 8 and 9 shows 3 bands with the 4.5 kb band from integration A. The integration C contains two bands of 3.3 and 3.5 kb.
The copy number can be determined by comparing the density of 3.3 kb band with3.5, 4.5 and 5.6 kb single copy bands.
#5, 6, 13 and 16 inherited
all the copies of the transgene.
#2 and 12 inherited integration A and B
#1,8 and 9 inherited integration A and C
#7 inherited integration A
# 4 and 11 inherited integration B
The various combinations of 3 integration sites indicate their genetic distance are far apart.