5w«i(i) E coRl (3647)

& /I (3668) pG am m a 2.0 EcoRl(*Xl668) EcoRl (3647) Sstl (3668) pG am m a 1.2 E c o R l(3647) ^5/11071(2429) 5 5 /1 (3668) p G am m a 1.0 EcoRl (3647) Sstl (2637) 55/1 (3668) Nsil EcoRl (3647) pG am m a 0.7 '^2974) (3668) E c o R l(3647) pG am m a 16 55/1 (3668) lact luo± hict

tra n sfe c tio n e ffic ie n cy and re p re se n t the level o f lu c ife ra se activity com pared with that seen with p G a m m a l6. The vector pG am m a 16 shows low activity when compared to all the other vectors and these values were comparable with those by pGL3- Basic. For this reason, here, the luciferase activity is com pared w ith th at seen with p G a m m a l6 in stea d o f the p ro m o te rle ss v e c to r .

The analysis of the transfection results presented in Figure 5.4 show s that the level o f tran scrip tio n d riv en by clones pGammaO.7 and pGam m al.O is basal (< 200 units o f luciferase) and has approxim ately equal levels in the facial m esenchym e, neural retina and dorsal root ganglia cells. It is clearly seen in these results that the high level of transcription o f the luciferase gene, occurs when clones containing prom oter inserts > 1.2 kb, are transfected into prim ary cell cultures o f neural retina. This h ig h le v e l o f tr a n s c r ip tio n (a p p ro x im a te ly 1 0 0 0 u n its of luciferase activity) in the retina is not p resen t in either facial mesenchyme or DRG cells.

The detailed analysis o f these results reveal several other less evident events, the level of transcription driven by clone pGammaO.7, c o n tain in g 700 bp o f the p u tativ e p ro m o te r, is higher than that o f the clone pGammal.O containing 1 Kb insert. T h is e v e n t w as c o n s is te n tly o b s e rv e d fo r at le a s t th re e in d ep e n d en t experim ents. p G am m al.O directs sim ilar levels of luciferase expression in all three cell types. It can also be seen that in the cells of the facial mesenchyme there is a decrease in

Figure 5.4: Luciferase activity o f the pGamma constructs fo llo w in g transfection into cultured cells isolated from Facial M esenchym e (A ), Neural Retina (B) and dorsal root ganglia (C ). V alues shown (mean o f three experim ents +- SD ) are n o rm a lised for tra n sfectio n e ffic ie n c y and represent the le v e l o f lu cifera se activity compared with that seen with pG am m al6, which was the same as that seen with pGL3-Basic.

A Facial M esen ch y m e 1500 1000 "3 5 500 - 200 vectors u 93 2 5 s

B.

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c . D orsal R oot G anglia 1500 = i 500

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luciferase activity as the clones becam e longer (e.g. luciferase activity is less with clone pG am m a3.7 then with pGammaO.7), and this decrease is gradual, with the exeption of pGam ma2.0.

Hardly any differences in expression levels occur when DRG cells are transfected with the same clones.

T h ese o b se rv atio n s su g g e st two p o ss ib ilitie s o f events: firstly , the 208 bp region contains sequences re c o g n iz e d by proteins present in the retina and therefore, these sequence acts as cell-specific enhancer element. The other possibility is that there are specific proteins in the facial m esenchym al cells and DRG cells that are able to recognize sequences and suppress gene expression. One event, however, does not exclude the other.

Although the DNA concentration was determined by O.D., I do not exclude the possibility of more copies of the gene being p r e s e n t in the tr a n s f e c tio n m ix tu r e o f pG am m aO .7 th an pG am m a3.7, and therefore the decrease o f luciferase expression fo r the facial m esen c h y m e as the c o n stru c ts h a v e lo n g e r upstream sequences, is not real.

In view of the dramatic and cell specific effect of the region betw een -1.0 and -1.2 on luciferase expression in neural retina cells, I focused on the activity of this region.

5 .2 .3 R ecom binant plasm id to express P -g a lac to sid a se gene

Schematically represented in the Figure 5.5 is the construct made, containing the bacterial Lac Z gene. p P g a l.2 p ro m o te r was transfected into facial m esenchym e and retina cell cultures as was previously described. This experim ent was to observe the capacity o f the 1.2 kb prom oter region to drive expression of a n o th e r gene than the lu c ife ra se and to see w h e th e r this expression was also specific.

Expression of Lac Z gene was revealed by the yellow colour display after the cell lysates had been assayed for presence of p -g a la c to s id a s e . T he ab so rb an c e (4 20nm ) e x p re ss e d fo r the transfected retina was 0.944 (+-0.104) and 0.325 (+-0.113) for the facial m esenchym e. In non-transfected cultures b ackground values were generally less than 0.1 0 0.

In order to see, in which cells does the 1.2 kb region drive specific expression, (e. g. neurons and not fibroblasts) in situ

s ta in in g o f p - g a l a c t o s i d a s e in c e l ls t r a n s f e c t e d w ith p p g a l.2promoter, remains to be done.

5 .2 .4 Construct containing 208 bp DNA fragm ent upstream the luciferase gene.

To determ ine whether the 208 bp fragm ent lying betw een -1208 and - 1 0 0 0 had p rom oter activity, rather than en h an cer

A.