EJECUCIÓN DEL PLAN ESTRATÉGICO

All of the methods mentioned thus far have the major drawback of a high false positive rate. In the case of differential display, the fact that the clones isolated are merely fragments of candidate genes often corresponding to the 3' UTR and not full length species further increases the potential work involved in the screen. In efforts to either refine existing strategies or to generate entirely new ways of differential

screening, many novel and interesting techniques have been tried some of which may appear to be of use to the mouse embryologist. Unfortunately problems exist with them all and no single technique has come to the fore.

1.5.1 Domain specific differential display

This involves reverse transcription with a short arbitrary primer followed by PCR with that primer and a degenerate primer homologous to a conserved region within a gene family e.g. zinc finger domains (Stone and Wharton, 1994). AP-PCR

parameters are generally employed. Some workers have carried out analogous studies using conventional differential display reverse transcription primers (Johnson et al,

1996). The process can be coupled with restriction analysis of the PCR products in order to further distinguish different members of the same family of genes which may give identically sized products (Fischer et a l, 1995). This approach can be viewed as a variant of PCR experiments with degenerate primers used to “fish” for genes with specific motifs except that only one specific primer is required. Differential clones can be sequenced and used with a degree of confidence in library screening.

1.5.1.1 Multiplex profiling

Multiplex profiling involves the reverse transcription of random primed cDNA with a mixture of several gene or domain specific primers in a single tube (Jensen et a l, 1996). As with domain specific differential display, an arbitrary primer is usually employed in combination. The reaction products can be visualised after

electrophoresis and differential species cloned, used for blotting and probed with

gene family specific probes or used in further differential display experiments as a form of enriched template.

1.5.2 Fingerprinting of 3* end cDNA fragments

Two strategies have been independently developed which aim to differentially fingerprint the 3' ends of cDNAs using a combination of restriction analysis and PCR. As in differential display, both methods attempt to reduce the complexity of the fingerprints obtained by either reverse transcription of only a fraction of all messages with anchored oligo dT primers or by prior restriction digestion of total cDNA with one of a panel of enzymes. However both have the potential advantage over

differential display of giving non-redundant information.

1.5.2.1 Differential 3' end cDNA profiling

This strategy was developed with a view to reducing the high false positive rates of differential display (Prashar and Weissman, 1996). A panel of two base anchored dT 18 primers is used which have long (20 base) 5' extensions to prime the synthesis

of double stranded cDNAs from the RNAs under study which are in turn digested with a restriction enzyme selected from a panel of 6-cutters. The cut cDNA is ligated to an adapter that has a 3' overhang for ligation and noncomplementary sequences at its 5' ends, thus giving it a Y-shape. The entire ligated cDNA is then PCR amplified at high stringency using a radiolabelled primer complementary to the 5' extension of the reverse transcription primer and an unlabelled primer complementary to the 5' end of the adapter. Only species which have the reverse transcription primer at their end will be seen after electrophoresis. Differentially expressed species can be

reamplified and cloned as in differential display and AP-PCR. The proponents of this technique claim less than 5% false positives and near total representation of all mRNAs using the combinations of reverse transcription primers and 6-cutter enzymes (S.Weissman Yale Medical School, personal communication). The published method requires at least lOpg total RNA.

1.5.2.2 Gene expression fingerprinting (GEF)

In this similar method, conventional cDNA synthesis is performed with

approximately 5|ig of each RNA using a single biotinylated oligo dT primer and digested with a frequently cutting restriction enzyme. 3' end directed restriction fragments are separated with streptavidin and ligated with an adapter to their restricted 5' ends. A PCR is then performed using an adapter primer and the

biotinylated oligo dT primer and after immobilisation of the products with

streptavidin the amplified cDNA is radiolabelled using the adapter primer and T7 polymerase. The immobilised radiolabelled cDNA can then be digested sequentially with a panel of restriction enzymes and released fragments electrophoresed in differential display gels (Ivanova and Belyavsky, 1995). Fingerprints for as many enzymes as possible can be made and the authors suggest that this will allow near total representation of all mRNAs. Differentially expressed species that are eluted from the gels are PCR amplified after homopolymer tailing with terminal transferase.

For both of these techniques large amounts of RNA were required in the original protocols. It may however be possible to modify them for an^ysis of PCR amplified total cDNA from dissected embryonic structures.

1.5.3 Combining subtraction and differential display

The term “subtractive display” has been applied to differential display performed on subtracted cDNA (Hakvoort et a l, 1994; Ariazi and Gould, 1996). The total cDNAs to be compared are tailed and PCR amplified prior to subtraction. The method of Ariazi and Gould entails cloning a fraction of the cDNA after each round of subtraction and PCR fingerprinting of each cloned aliquot with an arbitrary primer and a vector primer. The value of the subtraction step per se is not clear but the principle of differential display of PCR amplified cDNA is intriguing for the murine embryologist.

1.5.3.1 Representational difference analysis (RDA)

This is a PCR-coupled subtractive process based on a method for DNA difference enrichment that was initially developed in order to clone the differences between complex genomes (Lisitsyn et a l, 1993; Hubank and Schatz, 1994) (figure 1.2). The aim is to deplete the RNA populations being compared of common sequences and PCR amplify the differences. Double stranded cDNA is prepared from tester and driver RNA sources, digested with a 4-cutter enzyme and ligated with a

nonphosphorylated 12/24 adapter composed of one strand of 24 bases and the other of 12 bases. Only the 24-mer strand is covalently attached to the 5' ends of the cDNA and so the 12-mer species is melted off and the cDNAs are amplified with a primer complementary to the 24-mer. The adapter sequences are then removed from the cDNAs by restriction digestion and the amplified tester cDNA ligated with a different 12/24 adapter before a subtractive hybridisation is performed with a 100- fold excess of driver to tester. After the hybridisation the mixture is PCR amplified

Figure 1.2

Schematic representation of RDA (after Hubank and Schatz(1994)). Amplified tester and driver cDNA populations are derived by PCR as described in the text. After restriction digestion, a 12/24mer linker is ligated (shown in blue). As the linker is nonphosphorylated, only the 24mer component is ligated to digested cDNA. The cDNA is digested with a second enzyme and a second linker (shown in brown) ligated to the tester cDNA only. After hybridisation, the only cDNAs which can be amplified in a second round of PCR using primers complementary to the second linker are those which are enriched in the tester population. The resulting first difference product may be enriched by further rounds of hybridisation and PCR.

Tester cDNA ^ k. . Driver cDNA double stranded

# Restriction digestion (4 cutter)

Ligate 12/24mer

linker 24

12

g melt 12mer, fill - in, PCR amplify with ^ primer

complementary to 24mer

Tester representation Driver representation

Digest, ligate new 12/24mei _Digest