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D espite im provem ents to the exon trapping vector to create pSPL3 (Church et ah 199^, B urn et ah 1995 found that a large proportion (50%) of exon trapping products w ere artefacts derived from cryptic splicing in the HIV tat intron (see figure 3.2c). The sequences responsible for this cryptic splicing were rem oved greatly reducing the num ber o f such artefacts. Burn et ah (1995) also found that when am picillin resistant

BstXI half-site gen om ic D N A without exon

BstXI h alf-site

Splicin g

▼ C om p lete BstXI site

Figure 3.2 a E lim ination o f vector-vector sp lice products by BstXI d igestion

If no e x o n s are present in the gen om ic insert in the exon-trapping construct, v ector-vector sp lic in g occurs. In pSPLB, this brings together the tw o h a lv es o f a B s t X I site. B s t X I p red igestion prevents PCR am plification o f such sp lice products w h ich , b eca u se they are sm all, m ight have been am plified in preference to exo n -co n ta in in g products.

sp lice donor

site cryptic sp lice sites; acceptor in g en om ic seq u en ce, donor in vector intron

sp lice acceptor

L

Figure 3.2b BstXI digestion can rem ove som e cryptic sp licin g artefacts

A cryptic sp lice donor site within the FlIV intron is so m e tim es u tilised if the gen o m ic insert contains a cryptic sp lice acceptor site (Church et a i , 1994). pSPL 3 con tain s a B st XI site next to the m ultiple clo n in g site so that pred igestion w ith Bs tXI can prevent PCR am plification o f these cryptic sp licin g artefacts.

cryptic sp lice acceptor sp lice donor cryptic splice donor M CS sp lice acceptor

/

I

Figure 3 .2c BstXI predigestion does not prevent a second cla ss o f cryptic sp licin g A sec o n d set o f cryptic sp lice sites w ithin the H IV intron are so m e tim es u tilised , resultin g in the trapping o f H IV seq u en ces. pSPL 3 has recen tly been m o d ified to elim in ate these cryptic sp lice sites (Burn et a i , 1995).

cosm ids w ere subcloned into pSPL3, the use of am picillin to select for bacteria containing pSPL3 resulting in the selection o f recircularised cosm id vector fragm ents at the expense o f pSPL3-genom ic D N A constructs. R eplacing pS P L 3’s am picillinase gene w ith chloram phenicol acetyltransferase stopped selection for recircularised cosm id fragm ents. The new vector is called pSPL3B-CA M (Burn et al. 1995).

A nother problem o f exon trapping is that the exon trap products are often sm all and do not identify a large part o f the gene, so that the need to screen a cD N A library to get longer fragm ents negates the supposed advantage exon trapping has o f expression independence. E xon trapping products also make poor hybridisation probes for experim ents like screening cD N A libraries or Northern blot analysis because they are small. Tw o new vectors allow the subcloning of bigger genom ic inserts and the trapping of several exons o f a gene together. A.GET (Nehls et al. 1994) is a phage vector based on pL53In (Auch & Reth 1990) which will accom m odate inserts o f up to 19kb. sC O G H l (D atson et al. 1996) is a cosm id-based vector with an exon trapping cassette. It uses a m ouse m etallothionine-1 prom oter w hich drives transcription in m any different cell types, allow ing exon trapping in any cell type (it is possible that som e splicing occurs in a tissue-dependent manner) and a hum an grow th horm one intron.

There are also several m ethods of trapping of 5 ’ and 3 ’ term inal exons w hich are not easily trapped using conventional vectors as they only have one splice site. C onventional vectors m ay be used in com bination w ith 5 ’ or 3 ’ RA C E to trap term inal exons by using the g en e’s natural prom oter or polyadenylation site instead o f those from the vector. 5 ’ or 3 ’ RA C E can then be used to am plify the ends o f the transcript (see figure 3.3 and D atson et al. 1994, D atson et al. 1996). H owever, trapping term inal exons is m ore efficient if the vector is slightly modified: pTA G 4 (K rizm an & Berget 1993) has no dow nstream vector exon, splice acceptor site or polyadenylation signal, so can only trap 3 ’ term inal exons, and sC 0 G H 3 has no vector prom oter or upstream exon, allow ing trapping only o f products w hich include the 5 ’ term inal exon and prom oter region o f a gene (D atson et al. 1996).

sCOGHl