Efecto de las mutantes sobre la inactivación rápida y lenta

Chapter Two

2.1 GENERAL CONSIDERATIONS

All the protocols described in this chapter were conducted at room temperature unless otherwise stated. All the reagents used were obtained from Sigma unless otherwise indicated.

2.2 MOUSE STRAINS

Randomly bred CDl mice were used as a 'normal' strain and were obtained from Charles Rivers. A colony o f mutant mice, which is one o f a number o f alleles at the splotch

locus, which disrupts the transcription factor Pax3 (Epstein et al., 1991b), was established

in our laboratory in 1992. A heterozygous female, determined by the presence o f a white belly spot, arising on the C3H/101 background was obtained from the MRC Radiobiology Unit, Harwell, UK. This female was paired with a CBA/Ca male, and their offspring mated inter se to found a randomly bred colony.

2.3 GENERATION OF EMBRYOS FROM TIMED PREGNANCIES

Mice were housed with a 16 hour light and 8 hour dark cycle, with the midpoint o f the dark period at 1 am. For the generation o f C Dl embryos, males and females were placed together overnight. The next morning, females were checked for a vaginal copulation plug. Upon finding a plug, the female was assumed to be pregnant and midday o f that day was designated embryonic day (E) 0.5. Mating male and female Sp^^/+ mice

together generated litters containing +/+, Sp^^/+ and Sp^^/Sp^^ embryos in the standard

ration o f 1:2:1. Generation o f timed pregnancies was carried out in the same way as described for generating CDl embryos.

2.3.1

Dissection of embryos for in situ hybridization

When embryos were at the required developmental stage, the pregnant female was killed by cervical dislocation. The abdomen was washed with 70% industrial methylated spirits (IMS) (Hayman) in MilliRO water (deionised) and cut along the midline to expose the internal organs. The uterus was located underneath the intestines and detached from the two ovaries and the cervix, keeping both uterine horns intact. The uterine horns were then quickly placed in Dulbecco’s Modified Eagles Medium (DMEM (Gibco BRL)) containing 10% fetal calf serum (PCS). Individual conceptuses were dissected free o f the myometrium and decidua with fine forceps, and Reichert’s membrane was removed. The yolk sac was also removed, and in the case o f embryos, was retained and washed in phosphate-buffered saline (PBS), to remove contaminating maternal blood, and stored at -20°C for future genotyping. The remaining membrane, the amnion, was then removed and the embryos were washed in diethylpyrocarbonate-treated (DEPC) (see section 2.10.1) PBS on ice, fixed in 4% paraformaldehyde (PFA) in DEPC-treated PBS for 24-72 hours at 4°C whilst gently rocking. After fixation, the embryos were again washed in DEPC-treated PBS on ice and serially dehydrated through 30%, 50% to 70% IMS in DEPC-treated MilliQ water (ultra pure deionised water). At this point they were stored at room temperature or at 4°C until further use (see sections 2.4.2 and 2.4.3).

2.3.2

Dissection of embryos for immunohistochemistry

Embryos for use in immunohistochemistry were dissected and fixed using the same method used for preparation o f embryos for in situ hybridisation. DEPC-treated PBS,

however, was not essential, and a standard preparation o f PBS was used, as there was no need to protect against ribonuclease (RNAse) contamination (see section 2.10.1).

____________________________________________________________________________Chapter Two

2.4 GENERAL HISTOLOGY

2.4.1

TESPA-coating slides

To ensure tissue sections remain attached to the slide, a coating o f 3-aminopropyl- triethoxysilane (TESPA) was used. This reagent acts to electrostatically charge the slide, making the slide ‘sticky’ to the proteins within the tissue section. Slides were coated by immersion in 2% TESPA in acetone (BDH) followed by two brief washes in acetone, two brief washes in MilliRO water, and then left to dry overnight at 37°C. Two sets o f slides were prepared, one for protein studies, and the second for RNA work. This second set o f slides were prepared under RNAse-free conditions, baking the slides before coating and also using baked glassware and DEPC-treated MilliRO water, as described in section 2.10.1.

2.4.2

Wax embedding and sectioning

Embryos stored in 70% IMS in MilliRO water, were serially dehydrated through 85% to 95% IMS, followed by two washes in 100% ethanol (Hayman). The length o f time o f each wash was determined by the size o f the embryos. In general, solutions for embryos at E9.5 were changed after 15 minutes, embryos at E l0.5 after 30 minutes, embryos from E l 1.5 to E l3.5 for 60 minutes and embryos at E14.5 after 90 minutes. After dehydration, embryos were placed in Histoclear (National Diagnostics), a lipid solvent used to

penetrate the tissue o f the embryo. After three washes in Histoclear, embryos were transferred into a mix o f Histoclear and paraffin wax (Raymond Lamb) in a ration o f 1:1 at 62°C. Again, the time in wax was dependent on the age o f the embryo, as described above. Following this, embryos were placed in 100% wax at 62°C for three changes.

The wax acted to penetrate the embryo, replacing the Histoclear and stabilising the RNA and proteins within the tissue.

After the last change o f wax, embryos were orientated to provide transverse, sagital or frontal sections within the wax using heated needles to position the embryos and also keep the embryo in the correct position until the wax had solidified. Embedded embryos were sectioned on a microtome (Optech Instruments model HM 330) to 8

pm. Sections were floated on MilliRO water overlaid on TESPA-coated slides, and then heated to 40°C. The heating acted to flatten out the wax sections on the surface o f the water, enabling a smooth surface to adhere to the slide. The water was then removed and the slides were dried overnight at 37°C, and kept dry with desiccant until use.

2.4.3

Wax embedding and sectioning of embryos for in situ