DIRECCIONAMIENTO ESTRATÉGICO

IMR32

3T3

Spl

1A3) (Figure 3). H owever, low levels o f reporter gene expression are still observed in the non-neuronal M ^ n on-expressing c e ll line (3T 3) is not ab solu tely silen t. One p o ssib le explanation is that the elem en t(s) that silen ce expression o f the gene in 3T3 cells are not present in any o f the constructs used in this study, but exists further upstream or downstream. There are many exam p les o f d istal silen cin g elem en ts, for instance, silen cing o f L I-C A M gene expression is conferred by an RE-1 within the second intron (more than lOkb downstream o f the transcription start site) and by sequences in the first intron (Kallunki et al., 1997). A lternatively, the expression seen in 3T3 may be an artefact o f transiently transfected D N A . In som e ca ses, a region containing the prom oter and regulatory elem en ts id en tified by transient transfection assays in c e ll lin es for a particular gene appears to be sufficient to drive tissu e sp ecific expression o f a reporter gene in transgenic m ice. H ow ever, this is not alw ays the case, as has been shown for the 6 8-kDa neurofilam ent (NF-L) gene. It has been found that 1.7 kb o f sequence 5 ’ to the transcription start site o f the NF-L gene is capable o f driving expression o f a reporter gene in a tissue sp ecific manner in transgenic m ice, but is active in both NF-L expressing and n on -ex p ressin g c e ll lin e s in transient transfection assays (Nakahira

et al., 1 990). A lth ou gh transient transfection assays represent a very sim ple and u sefu l protocol to identify regulatory elem en ts, they have a number o f lim itations. One potential problem is the number o f cop ies of c ij-e le m e n ts introduced into the cell by transfection. D ue to a high copy num ber o f tr a n sfe cte d e le m e n ts, tran scrip tio n fa c to rs m ay b eco m e lim itin g and on ly partial e ffe c ts ob served. R eporter gen e ex p ressio n driven by the 1.7 kb fragment detected in NF-L non-expressing cells by transient transfection assays could be due to a de-repression effect due to lim iting amounts o f a factor required to repress transcription in these ce lls. H ow ever, stable ce ll lines produced using the same construct and

c o n ta in in g o n ly o n e c o p y o f th e r e p o r te r g e n e in te g r a te d in to ch ro m o so m a l D N A , s till sh o w ed ec to p ic e x p r e ssio n o f the reporter g en e (N akahira et al., 1990). In this case, it is p o ssib le that exp ression o f the NF- L g e n e is c o n tr o lle d by s e le c tiv e d e -a c tiv a tio n o f its prom oter during d e v e lo p m e n t, w h ich m ay in v o lv e m o d ific a tio n s o f th e D N A su ch as m é th y la tio n and o ccu r o n ly d uring a ce rta in sta g e o f d e v e lo p m e n t. T h e refo re, in tro d u cin g a prom oter that has n ot b een d e -a c tiv a te d , into c e ll lin e s that have already p assed this sta ge o f d ev elo p m en t, w ill a llow the prom oter to drive exp ression o f its gen e, or a reporter gen e, in a non­ s p e c if ic m anner. It w ill thus be im p ortan t to a n a ly s e any p u ta tiv e regu la tory reg io n id e n tifie d by tran sien t tr a n sfe ctio n in the co n te x t o f tr a n s g e n ic m ic e .

W ithin the region betw een - 3 7 2 and + 6 0 2 o f the M, gen e, there is a GC box at position +17 to +23, which is able to bind S p l in 3T3 cells and a 20 -2 5 kDa protein in IM R 32 c e lls (Figure 4 and 5). H ow ever, this elem ent d o es n o t appear to be required for the ex p r e ssio n o f the M i g en e in transient transfection assays in the c e lls an alysed (F igure 3). S p l p lays an im portant ro le in the con trol o f c e ll c y c le -r e g u la te d g e n e s and in the tr a n s c r ip tio n a l r e g u la tio n o f t is s u e s p e c i f i c and u b iq u ito u s g e n e s (review ed in (L ania et al., 1997)). It is lik ely that S p l acts in com binatorial m an n er w ith o th er tr a n sc r ip tio n fa c to r s , so th e p o s s ib ilit y o f the ex iste n c e o f an additional factor, with a binding site in a region o f the M, gen e further upstream or dow nstream o f the fragm en t a n a ly sed , acting in conjunction w ith S p l and/or the other factor id en tified to bind the GC box cannot be ruled out. In the case o f the M 4 gen e, a tandem o f three GC boxes w as fou n d to bind sev e ra l p rotein s, in c lu d in g S p l , but n o e f fe c t w as ob served w hen the GC b ox tandem w as d eleted (W ood et al., 1996). The potential fu n ction al role o f the Oct-1 and N F k B b in d in g e lem en ts present in the M, gen e rem ains to be determ ined. N F k B is found in the cytoplasm

b ou n d to the in h ib ito ry m o le c u le IkB . W hen IkB is p h o sp h o ry la ted , it r e le a s e s N FkB , w hich m o ves to the n ucleus to fu n ction as a transcription fa ctor (M ay and G hosh, 1997). D iv erse stim uli produce the release o f N FkB by Ik B in d iffe r e n t tis s u e s , in c lu d in g heat sh o c k , io n is in g ra d ia tion , lip o p o ly s a c c h a r id e and c y to k in e s (Basu et a l , 1998; D eshpande et a l , 1997; M araldi et a l , 1997; P ogliaghi et a l , 1995). It is not y et determ ined whether the N FkB elem ent binds NFKkB or w hether this elem en t has any fu n ction al ro le in regulatin g M i ex p ressio n . H o w ev er, the con struct pG L3 -3 7 2 /+ 1 6 9 M l w h ich co n ta in s the GC b o x , O ct-1 and N FkB site s did not drive ex p ressio n o f the reporter gen e in the M, exp ressin g c e ll lin e, IM R 32 (S ee Chapter 4 and Figure 6a), su g g estin g that th ese elem en ts do not have an e ffec t on Mi tr a n sc rip tio n .

CHAPTER 4