6. MATERIALES Y MÉTODOS
7.4 Deleción del péptido IFM
2.10.1 Treatment of glassware and solutions for use with RNA
protocols
All protocols described in this section were carried out in conditions designed to
eliminate contamination o f samples with ribonuclease (RNase). All glassware, including slides to be coated with TESPA, were baked at 180°C for eight hours to inactivate RNase. Sterile plasticware was used routinely. To inactivate RNase in aqueous solution, 0.5 ml/L o f DEPC was added to the solution and left overnight, and was then autoclaved the following day. DEPC is an inhibitor o f RNase, and is broken down into CO2 and ethanol by autoclaving. However, Tris and EDTA solutions, and solutions which could not be autoclaved, were made up in DEPC-treated bottles (those used to prepare DEPC-treated water), with DEPC-treated MilliQ or MilliRO water, and using solids that were kept separate from the general laboratory stocks, to reduce the likelihood o f RNase contamination.
2.10.2 Preparation of RNA probes for in situ hybridisation
To enable sense or antisense probe synthesis, plasmids containing the required cDNA insert, were linerarised by digestion with the restriction enzymes listed in Table 2. 4 and carried out as described below in section 2.10.2.1. A cDNA map o f each gene
investigated is shown in Figure 2.3. These maps indicate the region from which the fragments were cloned and, therefore, the region to which the labelled probes bind to during in situ hybridisation.
2.10.2.1 Restriction enzyme digest of plasmid DNA
Linearisation enables the RNA polymerase to access the DNA and prevents read-through by the polymerase into the vector. Restriction endonucleases recognise specific
sequences o f DNA and cleave the DNA at these sites. Restriction digest reactions were carried out for two hours in volumes between 20-200 pi with 1 pg o f plasmid DNA / 20 pi. Temperature and reaction buffers used varied according to the enzyme and were specified by the manufacturers (Promega or Gibco BRL). After digestion, the linerarised plasmids were purified by precipitation with 2x volume o f ethanol and 1/20 volume o f 4 M C2H302Na pH 4.5 at -20°C overnight or -80°C for 1 hour. The sample was then spun at 7500 g at 4°C for 30 minutes, the pellet washed twice in 70% ethanol in MilliQ water, resuspended in 10-100 pi o f TE and stored at -20°C.
2.10.2.2 Synthesis of digoxigenin-labelled RNA probe
Digoxigenin (DIG)-labelled RNA probes were synthesised by in vitro transcription using
a Boehringer Mannheim DIG RNA labelling kit. Total reaction volumes o f 20 pi contained: Ix transcription buffer, 10 mM DTT, 50 units o f RNAse inhibitor, 2 pi digoxigenin-labelling mix (10 pM ATP, 10 pM CTP, 10 pM GTP, 6.5 pM UPT, 3.5 pM DIG-11-UTP, pH 7.5), 1 pg o f linearised plasmid and 10 units o f the required RNA polymerase as listed in Table 2 .4 . The reaction was carried out at 37°C for two hours. To assess whether the reaction had been successful, 1 pi o f reaction mix was checked by agarose gel electrophoresis. A successful transcription should show, when the gel is illuminated under UV, a newly synthesised RNA band that is 10-20 fold brighter than the template plasmid DNA band. The synthesised DIG-labelled RNA probe in the reaction mix was then precipitated with 2x volume o f ethanol and 10 pi o f 4M LiCl at -20°C
Chapter Two
overnight. Probes were spun at 7500 g at 4°C for 30 minutes, the pellets were twiee in 70% ethanol in DEPC-MilliQ water, air-dried and resuspended in TE approximate eoneentration of 0.1 pg/pl, and stored at -20°C until use.
washed to give an
BMP’2 \BMP-4 Msx~l Msx-2 Tenascin Smad6
Obtained from Brigid Brigid Paul Paul Deborah Vicey
Hogan Hogan Sharpe Sharpe Henderson Reed
Vanderbilt Vanderbilt GK T Dental G K T Dental Institute o f Institute o f University U niversity Institute, Institute, Child Health, Child Health, M edical School, N ashville. M edical S chool, N ashville.
London London London. London.
Accession no. ^ .N M 007553 1 N M 007553 N M 0 1 0 8 3 5 ' m m m - : N M 0 1 1 6 0 7 ' # [ 0 0 8 5 4 2 .... Size, Kb 1.2 1.0 0.7 0.8 0.4 0.6 Vector pBSII ^SK pSP72 ps972 ps972 ' pGEM-T 1.pGEM-T easy Antisense: Endonuclease
Xbal EcoRI Bglll Hindlll Sstll Apal
Antisense: j ' Polymerase |
T3 Sp6 Sp6 ; ;T7 Sp6 ‘ .j Sp6
Sense:
Endonuclease
Xhol Smal EcoRI Bglll
...
SstI SstI
Sense: ;T7 T7 T7’ T7
Polymerase
Table 2. 4 In situ hybridisation probe details
Figure 2. 3 cDNA maps o f BMP-2, B M P -4,, Msx-1, Msx2, Tenascin-C and Smad6
(A-F) cDNA maps created from the public data in GenBank. The accession numbers fo each gene are listed in Table 2 .4 . The numbers underneath each cDNA map indicate th length o f the cDNA in base pairs (bp). The legends for the functional domains are included in each map.
Due to the inclusion o f the homeodomain within the Msx-1 probe (C), the specificity o f this probe needed to be addressed (compare C and D). In addition to the data shown in this thesis, where distinct expression patterns between Msx-1 and Msx-2 i
observed (compare sections 3.3.3.1 and 3.3.4.1), specificity has also been demonstrated previously by Jowett et al, 1993 and Phippard et al, 1996. Using identical probes for
Msx-1 and Msx-2, to those used in this thesis, in situ hybridisation analysis during mous tooth (Jowett et al, 1993) and mammary gland (Phippard et al 1996) development has shown distinct spatial and temporal expression patterns between the two genes. These data suggest that the Msx-1 and Msx-2 probes generated by Paul Sharpe, used both with this thesis and by Jowett et al, 1993 and Phippard et al, 1996 amongst others, are indeed gene specific. D) The Msx-2 probe is a double insert o f the region o f the cDNA indicate on the map, represented by a double black line.
Chapter Two 200 400 600 A) BMP-2 cDNA 1000 1200 1400 1600 bp I I Coding region
■
TG Fp pro-peptide region TG Fp-like domain Probe sequence (270-1470bp) Non coding sequence200 400 600 B) BMP-4 cDNA 1000 1200 1400 1600 bp I I Coding region H B TG Fp pro-peptide region TG Fp-like domain Probe sequence (~l-1000bp) sequence not in GenBank
300