Biology Reference
In-Depth Information
3.1.4. Freezing Mammary Rudiment for Transport or Storage
1.
As mentioned in Note 4 , it is highly preferable to transport dissected mammary
rudiment frozen. It may also be necessary to keep the gland frozen until a suitable
recipient is available. To do this, the tissue should be placed in a cryovial con-
taining 0.5 mL freezing mix and frozen slowly to -80
°
C ( see Note 8 ).
2.
Liquid nitrogen should be used for longer term storage.
3.1.5. Recovery of Frozen Tissue
1.
Frozen tissue should be rapidly thawed and washed several times in serum-free
medium (e.g., L15) to remove any traces of serum or DMSO before transplantation.
2.
Thawed tissue should be kept in serum-free medium on ice until transplantation.
3.2. Transplantation of Mammary Tissue into Recipient Mice
3.2.1. Breeding of Recipient Mice
To avoid tissue rejection, transplantation should always be into syngeneic-
recipient animals. Nude mice can be used as recipients, however, their mam-
mary glands are small and postoperative infections may cause problems. Most
transgenic animals are a cross between C57BL6 and 129 strains of mice and,
therefore, F1 progeny of 129xC57BL6 are ideal recipients ( see Note 9 ).
3.2.2. Clearing the Fat Pad and Transplantation
1.
Recipient mice must have their endogenous mammary epithelium removed by 21 d
after birth. As this is normally the time of weaning from the mother, it is usual to
wait until 21 d before operating. The #4 or abdominal glands are the easiest to
clear for transplantation. Clearing the fat pad can be done at the same time as
transplanting tissue and this is preferable because the animal then only undergoes
one operation. However, if this is not possible for logistical reasons, the fat pad
can be cleared and the animal left until required for transplantation.
2.
We routinely use 21-d-old F1 progeny C57BL6x129 mice which are quite skit-
tish. To minimize the stress of an intraperitoneal (ip) injection, the mice are sub-
dued in an anaesthetic box perfused with halothane, N 2 O, and O 2 before ip
injection of 10
µ
L/g body weight anaesthetic.
3.
Once anaesthetized, the abdomen is shaved and swabbed with a small amount of
chlorhexidine solution.
4.
The mouse is then restrained on its back on a cork dissecting board using small
elastic bands tied in a slip knot around each paw and secured at the other end with
a small pin.
5.
A small Y-shaped incision is then made in the ventral skin from just under the rib
cage to slightly down each of the hind limbs using eared scissors. If any small
blood vessels are accidentally nicked they are immediately cauterized to mini-
mize blood loss.
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