such as "Introduction", "Conclusion"..etc
H253 strain mice , ubiquitously expressing an X-linked nLacZ transgene (abbreviated to XLacZ), were obtained from the MRC Mammalian Genetics Unit, Harwell, UK. Hemizygous XLacZ+/- females (produced from crosses between XLacZ+/Y, H253 males and (C57BL/6 × CBA/Ca)F1 females) show mosaic transgene expression, after X-gal staining for β-gal
reporter activity, due to random X-inactivation during development.
Mice of the transgenic reporter lines Y001deltaDRR and Y223 (showing
mosaic expression of PAX6-GFP) were used to visualise stripe
dynamics by time-lapse confocal microscopy. The Y223 line carries 6
copies of a yeast artificial chromosome (YAC) containing the human PAX6 locus 
in which PAX6 protein expression is prevented by a GFP reporter gene
inserted into the PAX6 ATG start codon. In addition, the downstream
regulatory region (DRR) of the PAX6 locus is flanked by loxP sites.
Line Y001deltaDRR was produced by crossing Y001 (containing a single
copy of the above YAC with a 10–20 kb truncation) with the CAGGS-Cre line  resulting in germline deletion of the DRR region and leading to reduced GFP expression in the lens .
Whole eyes dissected at 3 to 52 weeks after birth were fixed and stained for β-gal reporter activity as described previously .
To prepare whole-mounts of the ocular surface, the eye was first
enucleated with the eyelid intact, the posterior segment and lens were
then removed and several radial cuts were made in the cornea and
conjunctiva to allow the tissue to be flattened. Where sections were
required the lens was removed before wax embedding. 7 μm
paraffin sections were cut on a microtome and mounted onto standard
microscope slides. The sections were first dewaxed in Histoclear (2 × 5
mins) and then dipped in 95% (vol/vol) ethanol (1 min). Sections were
then counterstained by dipping in Eosin (Surgipath) for 1 min, washing
in tap water (5 mins) and staining in 1% (wt/vol) Neutral Red solution
(3 mins). Slides were then rinsed in distilled water (2 × 1 min),
dehydrated by dipping in a graded ethanol series (70%, 95% and 100%
vol/vol) followed by xylene (2 × 1 min). Cover slips were mounted with
DPX mounting medium (VWR, Poole, UK).
As described previously 1 mm diameter wounds through the full epithelial thickness were made in XLacZ+/- corneas.
Eyes were then cultured for 24 hours and stained with fluorescein at 0,
6 and 24 h to visualise and photograph the wound margin .
Eyes were returned to culture after the examination at 6 h and after 24
h they were fixed and stained with X-gal as described previously .
For preliminary evaluation of different transgenic lines, GFP
expression in the corneas was imaged using a Leica MZFLIII fluorescence
stereo microscope fitted with a Coolsnap cf colour CCD camera
(Photometrics Ltd, Tucson, AZ). Eyes from line Y100deltaDRR were
wounded and enucleated in the same way as XLacZ+/- eyes. They were then cultured in a controlled environmental chamber (37°C; 5% CO2 in
air) surrounding the stage of a Leica inverted confocal microscope.
During culture the eye was supported on a specially designed stand so
the cornea faced downwards. Z-stack images were collected at intervals
over a 24 h period using a ×5 objective. Confocal images were analysed
using ImageJ, a shareware image analysis software package [47,48]. The 'Stackreg' plug-in 
developed by Philippe Thévenaz, (Biomedical Imaging Group, Swiss
Federal Institute of Technology Lausanne, Switzerland) was used to
correct for any movement of the eye between time points. The distance
moved by the leading edges of individual stripes were measured in two
ways. For method A, the distance moved in a straight line was simply
measured between the first and last time points in the experiment. For
method B, stripes were analysed by manually tracking and measuring the
distance moved between time points. Both methods used the 'MtrackJ'
plug-in developed by Erik Meijering (Biomedical Imaging Group
Rotterdam, Erasmus MC – University Medical Center Rotterdam, The
For analysis of variegated, mosaic patterns, it is important to
distinguish between different types of patches and clones. We use three
terms defined previously [25,50-53]. A patch is a group of cells of like genotype (or phenotype) that are contiguous at the time of consideration. A descendent clone is any group of clonally related cells irrespective of whether they have remained contiguous throughout development. A coherent clone is
a group of clonally-related cells that have remained contiguous
throughout development. The number of coherent clones per patch depends
partly on the proportions of the two cell populations in the mosaic.
For one-dimensional mosaics (strings of two cell populations) the
number of coherent clones per patch of cell population 'A' is estimated
as 1/(1-p) (where p is the proportion of cell population 'A' in the mosaic) [25,28,53].
The observed mean width of β-gal-positive stripes in the corneal epithelium was corrected for the probability that stripes would contain multiple adjacent β-gal-positive corneal epithelial clones. This involved dividing the observed mean width by the function 1/(1-p), where p is the proportion of β-gal-positive cells around the circumference [4,25,52,53]. For radial stripes that form a complete circle, the corrected mean stripe width is identical for β-gal-positive and β-gal-negative stripes because the numbers of β-gal-positive and β-gal-negative stripes are identical and the proportions of β-gal-positive and β-gal-negative
cells around the circumference are not independent. The reciprocal of
this corrected mean stripe width, expressed as the proportion of the
circumference, is the corrected stripe number. This provides an
estimate of the total number of corneal epithelial coherent clones
(both β-gal-positive and β-gal-negative) per circumference [4,5]. This estimate assumes (1) coherent clones are equal in size, or their sizes are normally distributed, and (2) β-gal-positive and β-gal-negative
coherent clones are randomly distributed around the circumference. Even
if these assumptions are not correct the corrected stripe number should
be proportional to the number of coherent clones so its application as
a comparative measure is still valid.
The clonal analysis of striping patterns between 15 and 52 weeks was carried out as described previously [4,5].
To determine whether stripe patterns varied around the circumference,
mean corrected stripe numbers were estimated separately in four regions
as well as the whole circumference for 15 mosaic eyes (from 23–24 week
old XLacZ+/- mice). Each region was equivalent to
half of the circumference (nasal or temporal; superior or inferior).
The nasal conjunctival epithelium was sutured to mark its orientation
and eyes were removed and stained. Stripes were counted and measured
separately in different hemispheres in two ways: nasal versus temporal
and superior versus inferior. Stripes on the nasal-temporal or
superior-inferior boundaries were included in both regions. To correct
for overlapping stripes, the corrected stripe number per region was
calculated by dividing half the circumference by the corrected mean
stripe width separately for each of the four regions.
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