Biology Reference
In-Depth Information
Tissue should be harvested around 8 wk after initial transplantation, cut into small
pieces, and then used to repopulate more host mammary gland. Serial transplan-
tation of mammary tissue is only successful for a limited number of generations
because of cellular senescence. The proliferative potential of normal mammary
cells declines with serial transplantation and is lost after 5-6 serial transplants
(11)
.
2.
It is possible to mate the recipient mice and thereby examine development of the
transplanted tissue. However, it should be remembered that because the ductal
network does not connect with the nipple, a fully lactational phenotype will not
be achieved as accumulation of milk will induce immediate involution of the
glandular epithelium.
3.
Determining the appropriate embryonic age at which to isolate mammary tissue
depends on several factors. First, the viability of the embryos; a phenotype which
is lethal at, e.g., embryonic day 15 (E15) requires tissue to be harvested before
that time. Second, the ease of sexing the embryos: from around E14 onward,
viable mammary tissue can only be isolated from females (G. Cunha, personal
communication). And third, the ease of finding the nipples on the developing
skin. These last two factors must be traded against each other. The difference in
anogenital distance, which is the main aid to determining the sex of the embryos,
is more obvious in larger embryos. However, at later stages of embryogenesis,
the developing hair follicles in the skin cause the formation of small bumps which
can be hard to distinguish from nipples. I have found F1 embryos (C57BL6x129)
of E16.5 to E17.5 to be the easiest to isolate mammary tissue from.
4.
Shipping embryos markedly reduces the viability of the mammary tissue. It is
therefore preferable to be able to isolate the mammary tissue from the embryo
immediately and process it for cryopreservation if transportation is required. This
is most easily done by travelling to the site of the transgenic mouse colony and
performing the tissue isolation there.
5.
Sexing mice by anogenital distance is a standard animal husbandry technique
used on newborn mice. Although the distances are smaller in embryonic mice, it
may be useful to have the differences between the sexes at birth pointed out by an
experienced animal technician.
6.
The #1 mammary glands lie just above the submandibular salivary glands and
great care should be taken when isolating the mammary rudiments not to take
any salivary tissue unintentionally as this will also transplant successfully. The
salivary glands can easily be distinguished in whole mounts (and even unstained
under the dissecting microscope) by their lung-like lobular appearance.
7.
In embryos with a skin detachment phenotype, the skin should not be lifted away
from the body of the animal. Instead the scissors should be used to cut into the
skin around the nipple to remove the gland. This phenotype applies to
6 integrin
null animals and may also be apparent in some mice with altered expression of
basement membrane proteins.
α
8.
We achieve slow freezing using a Nalgene “Mr Frosty” tub containing isopro-
panol which cools at 1
°
C per minute (
see
Subheading 2.1.
).
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