Modelación de la interacción genotipo X ambiente en rendimiento de híbridos de

Whole mount in situ hybridization was employed to determine tempo-spatial pattern of Cats expression during mouse embryogenesis. Mouse embryos were prepared and hybridized by standard protocols according to Wilkinson, 1992; with some modifications.

2.2.19.1 Preparation of mouse embryo

BALB/c mice (2.1.9) were paired and the females checked the following day for the presence of a vaginal plug, indicating 0.5 days post coitum (d.p.c) embryos. On the 9.5-14.5 d.p.c, the pregnant female mice were dissected, embryos were collected and placed in cold PBS-DEPC. Extra-embryonic membranes were removed and embryos were fixed in 4 % PFA (in PBS- DEPC), rocking O/N at 4°C. Next day embryos were gradually dehydrated (on ice) through 25 %, 50 %, 75 % methanol (in PBS-DEPC) for 10 min each and bleached for 1 hr with freshly prepared 6 % H2O2 (in methanol), Treatment with H2O2 has been found to decrease

considerably the amount of non-specific background. After bleaching, embryos were further dehydrated twice for 5-10 min in large amount of 100 % methanol and stored in this solution at -20°C. Treatment and storage of mouse embryos were carried out in scintillation vials. Embryos dissected for RNA extraction purpose (2.2.9), were stored at -80°C (in 1.5 ml tube) immediately after removal of extra-embryonic membranes and PBS-DEPC wash.

2.2.19.2 DIG-labeling RNA

RNA probes labeled with Digoxigenin-UTP were synthesized by in vitro transcription of Cats cDNA templates. The DNA to be transcribed was first cloned into pGEM®-T Easy (2.1.11) which contains promoters for SP6 and T7 RNA polymerases (Figure 2.1). Prior to transcription, enzymes that created a 5’-overhang were used in a digestion reaction (2.2.2.1) to linearise the template avoiding transcription to continue over the insert onto the plasmid itself. Digestion was carried out for 2 hrs with 10 µg DNA template in a 30 µl reaction. Linearised DNA was then purified using a QIAquick® nucleotide removal column (Qiagen) according to manufacturer’s instructions and eluted in 32 µl Ampuwa® H2O.

PI clone (850 bp) was linearised with SalI and transcribed with T7 RNA polymerase to produce anti-sense probe and linearised with NcoI and transcribed with SP6 RNA polymerase to produce sense probe. PIII clone (300 bp) was linearised with NcoI and transcribed with SP6 RNA polymerase to produce anti-sense probe (Figure 2.1).

2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800

Exon 2 Exon 3 Exon 4Exon 5Exon 6

P I

T7 RNA… SP6 RNA pol promoter

mCATSF113 mCATSR942 NcoI (3823) SalI (2921) 100 200 300 400 500 Ex3 Ex2 PIII NcoI (37)

SP6 RNA pol promoter

mCATSF364 mCATSR630

Fig. 2.1: PI and PIII clones used to produce sense and anti-sense WISH probes. PI and PIII clones were generated by RT-PCR from thymus RNA using primer pairs mCATSF113-mCATSR942 and mCATSF364– mCATSR630, respectively and by subsequent cloning of the 850 and 305 bp products into the pGEM®_{-T Easy}

vector. PIII was linearised with NcoI and transcribed with SP6 RNA polymerase to produce anti-sense probe and PI was linearised with NcoI and transcribed with SP6 RNA polymerase to produce sense probe. Arrows indicate binding position of CATS primers.

For the in vitro transcription the following components were placed in a 1.5 ml tube. 10 µl Template DNA (PI or PIII) prepared as described above 3 µl Ampuwa® H2O

2 µl TS-buffer 2 µl rNTPs DIG-Mix 1 µl RNase inhibitor

2 µl RNA polymerase (T7 or SP6)

Reaction was incubated for 2 hrs at 37°C. After that, 2 µl DNase I was added to the reaction, which was incubated for further 15 min. Transcribed RNA was then precipitated by adding 400 µl 100 % ethanol, 1 µl tRNA (20 mg/ml) and 33 µl 7.5 M NH4Ac, gently mixing and

incubating for 1 hr at -80°C. RNA was centrifuged at 15 000 rpm for 30 min at 4°C, pellet was washed with 70 % ethanol (15 000 rpm, 15 min, 4°C) and resuspended in 100 µl Ampuwa® H2O plus 1 µl RNase inhibitor. Quality and yield were checked, by loading 5 µl of

2.2.19.3 Hybridization of mouse embryos

Embryos prepared as described above (2.2.19.1) were re-hydrated through 75 %, 50 % and 25 % methanol (in PBS-DEPC) incubation for 10 min each. Re-hydrated embryos were then washed for 10 min with PBT and transferred to the slots of the InsituPro robot (ABIMED), where an automated hybridization took place. In contrast to manual hybridization, the apparatus allowed rapid changes of solutions and constant temperature throughout all steps. All buffers used were listed in 2.1.5. Briefly, embryos were washed twice with PBT for 10 min. For embryos older than 10.5 d.p.c., 5 min treatment with proteinase K buffer was needed. Proteinase K was used to improve the signal by increasing the accessibility of target RNA. After proteinase K treatment, embryos were washed twice with 2 mg/ml glycine in PBT (10 and 5 min, respectively), then twice with PBT. Prior to pre-hybridization, embryos were treated with RIPA-DEPC and 4 % PFA/ 0.1 % GA (10 min and 20 min, respectively), with two PBT washing steps (10 min) between and after treatments. Pre-hybridization was carried out with hybridization solution (WISH) plus 100 µg/ml tRNA for 3 hrs at 70°C. For hybridization, DIG-labeled RNA probe was previously denatured and then added to the same solution diluted 1:100 dilution. Hybridization was carried out for 16 hrs at 70°C. Post hybridization washes were performed to remove unbound probe. Firstly, embryos were washed for 1 hr (4 x 15 min) with hybridization solution (WISH) free from RNA probe, at 70°C. Then treated with 100 µg/ml RNase A in TNT for 1 hr at 37°C. RNase A treated embryos were washed altogether for 5 hrs (5 min, 3 x 10 min and 9 x 30 min, respectively) in SSC/FA/ Tween® 20 at 70°C, then for 20 min in TBST and MABT (2 x 10 min, each) at RT. Before incubation with antibody, embryos were blocked with 2 % blocking reagent (Roche) in MABT for 3 hrs. Meanwhile a 1:5000 dilution of Anti-Digoxigenin-AP conjugate (Roche) was pre-incubated with 1 % blocking reagent (Roche) in MABT for 1 hr at 4°C with gentle rocking. Immuno-hybridization was carried out for 12 hrs in the AB solution described above. Immuno-hybridized embryos were washed altogether for 14 hrs with TBST (2 x 5 min, 7 x 30 min and 10 x 1 hr, respectively). After washing, embryos were transferred to Cryotube™ vials, washed with NTMT (2 x 5 min) and stained using centrifuged BM purple AP substrate (Roche) O/N at 4°C. The staining reaction was kept in dark and the color development was monitored. When the desired extent was achieved, reaction was stopped and embryos were fixed in 4 % PFA for 1 hr at RT. Stained embryos were stored in the same solution for several month. For documentation, embryos were transferred to PBS and mounted on a thin layer of agarose (in Petri dishes) under a MZ APO stereomicroscope (Leica) in dark field illumination

camera using a Leica QWin software (Leica Microsystems Imaging Solutions, Cambridge, UK) and pictures were adjusted for brightness and contrast in Adobe Photoshop® 7.0 (Adobe Systems, Mountain View, U.S.A).

2.2.19.4 Dissection of hybridized mouse embryos and microscopy

Mouse embryos hybridized and stored as described above (2.2.19.3) were incubated in 30 % sucrose solution (in PBS). After having sank to the bottom of the tube, embryos were incubated in 7.5 % gelatine solution (in 30 % sucrose solution) for 2 h at 42°C in a water bath. In a Petri dish, a thin layer of the 7.5 % gelatine solution was kept at RT until became solid and embryos were then embedded with the same solution onto this layer. After O/N incubation at 4°C, embryos were recovered into small gelatine blocks. Gelatine blocks were placed in alu-folio boxes (1 x 1 cm), which were filled with Jung Tissue Freezing Medium™, frozen in dry ice and stored at -80°C. Embedded embryos were sectioned in 35 µm slices with a Cryotome CM 1850 (Leica) at -30°C and placed onto pre-cooled slides. Cover slips were mounted on the slides with Kaisers Glyceringelatin and stored at RT. Sections were observed under an automated Axioplan 2 Imaging microscope (Carl Zeiss, Jena, Germany) equipped with an AxioCam HR digital camera. Images were recorded using the AxioVision 3.1 software (Carl Zeiss, Jena, Germany) and pictures were processed as in 2.2.19.3.