Testing transgenic regulatory elements through live mouse imaging.
To overcome positional and methylation effects on transgene expression, we developed a universal cloning cassette for in vivo assessment of regulatory elements using the luciferase reporter gene and the CCCD camera. Monitoring luciferase expression pattern in live mice enables screening of large numbers of transgenic founders quickly and inexpensively. We demonstrate that in the engineered transgenic mice, the chicken beta-globin 5'HS4 insulator did not always provide the desirable expression pattern, and the Island Element, responsible for the demethylation of the surrounding DNA region, was not beneficial. Both tested liver-specific and developmentally regulated promoters exhibited the expected expression pattern in most transgenic founders.
Goldyne Savad Institute of Gene Therapy, Hadassah - Hebrew University Medical Center, P.O.B. 12000, Jerusalem 91120, Israel. Naamitk@gmail.com
Naama
N
Klopstock
Carol
C
Levy
Devorah
D
Olam
Eithan
E
Galun
Daniel
D
Goldenberg
D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D019175:DNA Methylation; D005786:Gene Expression Regulation / Q000502:physiology*; D005914:Globins / Q000235:genetics / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D038101:Insulator Elements / Q000502:physiology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D018715:Microscopy, Video; D009928:Organ Specificity / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology*; D019076:Transgenes / Q000502:physiology*
D005914:Globins; D008156:Luciferases