Figure 1.
Expression of αA-crystallin driven fluorescent proteins in α-TN4 mouse lens epithelial cells.
(A) Schematic map of αA-crystallin fluorescent protein constructs drawn to scale. Features include the 2.5 kb αA-crystallin promoter and the 1 kb fluorescent protein coding sequence and polyadenylation signal (1.6 kb for tdTomato). (B) Fluorescent protein expression 48 hours post-transfection. Images were taken in grayscale and pseudo color added using AxioVision digital image processing software.
Figure 2.
Fluorescent eye marker visibility and variability.
(A) Fluorescent proteins expressed in the lens can be visualized under a handheld flashlight. All five fluorescent eye markers can be seen using the GFP flashlight while only mOrange, tdTomato, and mPlum can be seen using the RFP flashlight. (B) EGFP and tdTomato marker expression varies between transgenic lines (Low, Medium, High). Inset shows EGFP “high” line is visible in room light with no excitation. EGFP “medium” and tdTomato “high” transgenic lines are also shown in pigmented (Agouti, Black) backgrounds. Note that the visibility of fluorescent eye markers is reduced in pigmented backgrounds.
Figure 3.
Co-segregation and co-integration of fluorescent eye marker and transgene.
(A) tdTomato fluorescent eye marker co-segregates with and accurately marks the presence of an unrelated transgene (Luc-PB[mut]7). One litter of pups numbered 1-7 is shown. The unrelated transgene was amplified by PCR (upper panel) and the tdTomato marker was visualized under the GFP flashlight in room light (lower panel). (B) Schematic drawing of PCR strategy. One PCR primer (small arrows) was designed against the 3′ end of the Luc-PB[mut]7 transgene (gray box) and the other primer against the αA-crystallin promoter (white box) driving the tdTomato marker transgene. (C) The hybrid PCR product from the transgene concatamer is present in two transgenic animals (+) but not wildtype littermates (−).
Figure 4.
The intensity of the fluorescent eye markers allows differentiation of heterozygous from homozygous transgenic mice.
(A) Heterozygous and homozygous adult mice carrying mCFP or tdTomato markers can be easily identified under a handheld flashlight. (B) Homozygosity in adult mice can also be determined more quantitatively using whole animal in vivo fluorescence imaging. (C) Neonatal mice carrying EGFP or tdTomato markers can also be easily identified as heterozygous or homozygous under a handheld flashlight. (* marks heterozygous animals, # marks homozygous animals)
Figure 5.
Fluorescent eye markers allow visual identification of double and triple transgenic mice.
(A) Double transgenic mice carrying the EGFP marker and either tdTomato or mPlum can be easily distinguished from single transgenic littermates under the GFP flashlight. (B) Single, double and triple transgenic mice can be identified using mCFP, mOrange and mPlum eye markers. All six possible combinations are distinguishable under the GFP flashlight. Insets show close up view of fluorescence in the eyes of double and triple transgenic mice.