2.1. Animal and tissue handling

All animals were originally derived from the same transgenic mouse line [10], [11]. They are both null for murine TTR and homozygous for the ATTRV30M transgene but on two different genetic backgrounds; the 129×1/SvJ background (as of now shall be referred to as: TM 129) and the 129×1/SvJ/C57BL/6J hybrid outcross background (as of now shall be referred to as: TM 129/BL6). Two 129×1/SvJ and two C57BL/6J animals of the appropriate age group, depending on the application, were used as wild type control animals.

The mTTR^−/−hTTR^V30M/V30M mice were generated from a single pair of mTTR^−/− hTTR^V30M mice (of 129×1/SvJ background, obtained from M. J. Saraiva) that were crossed amongst them to generate mTTR^−/−hTTR^V30M/V30M pups. These were then inter-crossed to generate the colony of experimental mice. The number of copies of the V30M transgene was monitored by real time PCR. These mice are referred to as TM 129.

To generate the TM 129/BL6 colony, male mice mTTR^−/−hTTR^V30M/V30M (in 129×1/SvJ background) were crossed to C57BL/6J females in order to generate F1 mice mTTR^+/− hTTR^V30M. These were then inter-crossed to generate F2 mTTR^−/−hTTR^V30M/V30M mice which were then bred to generation 7 through successive brother-sister mating. These F7 progeny were used to produce the experimental mouse line which was used throughout the experiments. Specifically, the F7 generation we used was made up of 2 males and 6 females. Their progeny is considered to be of mixed genetic background, segregating alleles of 129×1/SvJ and C57BL/6J origin.

Real time PCR was used to ensure that all animals included in the experiment had the same copy of transgenes (Fig. 1A). Furthermore reverse transcription real time PCR was used to confirm that RNA expression for human TTR was similar in liver tissue of the two backgrounds used in the experiments (Fig. 1B).

(A) Real-time PCR results of the number of transgene copies found in the animals of TM129 pure background and TM129/BL6 mixed background, (B) Reverse transcription real-time PCR results from the liver tissue of TM129 pure background and TM129/BL6 mixed background animals measuring the amount of human TTR RNA expressed in liver, (C) Western blot of TM129 stomach samples from different mice and a single wild type 129 animal for hTTR and GAPDH. The strong TTR band seen consists of monomers.

Three groups of animals were used from each background; group A (3–6 months), group B (9–12 months) and group C (15–18 months). Each TM 129 age group was comprised of 15 mice (7 M/8 F), whereas each TM 129/BL6 group was comprised of 10 mice (5 M/5 F) each. All animals were kept in a regular 12 h light-12h dark cycle and were given free access to water and food, under SPF conditions. Animals were separated in cages depending on the age group they were assigned to and their sex. All animal involving experiments were carried out in accordance to the 86/609/EEC Directive.

Mice were anesthetised and then euthanized using Tribromoethanol (Avertin) though IP injection at a dose of 250 mg/Kg. The animals were then exsanguinated via PBS perfusion to reduce the contribution of plasma in the measurements. Tissues were obtained at the required time points. Tissues were processed for immunohistochemistry by carrying out overnight 4% PFA fixation followed by wax embedding or were frozen and kept at −80 °C for western blot analysis.

Amyloid deposition assessment was confined to stomach tissues since this organ is heavily involved in amyloid deposition at an early age in this model of ATTRV30M neuropathy.