ENRIADO SIN MACHACADO PREVIO 1 Extracción de las fibras

Several attem pts to transfect RBL-2H3 cells were m ade using this method. Initially approximately 2x10® cells were used each time b u t since the efficiency of transfection Is only 1 In 10® a t best (Dr. Birgit Helm, personal communication), cell num bers were Increased.

Subsequent attem pts using this method were more encouraging. Using high cell num bers of approximately 5x10^ per attempt, approximately 2 0 transfected colonies per

dish reached the 20-30 cell stage. However In repeated experim ents all colonies Inexplicably died a t th e sam e time. Eventually a faulty Incubator was found to be the

source of th e problem. Thus, a batch of identically treated pNV-XNA2-transfected RBL- 2H3 cells cultured in a different incubator yielded 20-30 stably-transfected colonies per dish. Most of these were isolated and further expanded in 96-well plates prior to

tran sfer to duplicate 30mm dishes. One dish of each of 26 stable pNV-XNA2

transfectants, when confluent, was iysed directly into 250|il of SDS-PAGE sam ple buffer for analysis of annexin II content. lOOpl of each lysate was subjected to SDS-PAGE, W estern blotting and immunoblotting with anti-annexin n antibody HH7. The resu lts of these are shown in the top two panels of figure 5.3, the left h an d track in each panel is control RBL-2H3 cell lysate. None of these clones showed a significant alteration in the level of expression of annexin II. (Uneven fading of the blots prior to photography h a s accentuated the differences seen in figure 5.3.)

From further rounds of transfections, 32 pNV-XNA2 colonies and 34 pNV-ANX2 transfected colonies were isolated and cultured. A 3cm dish of each w as lysed into 250pl of lysis buffer, centrifuged to pellet cell nuclei and the su p ern ata n t mixed with an equal volume of 2xSDS-PAGE sample buffer. lOOpl of each sam ple w as electrophoresed and immunoblotted for annexin II with HH7. None of the pNV-XPJA2 transfected cells showed a significant reduction in annexin II levels (central three panels of figure 5.3); and none of the pNV-ANX2 transfected clones showed a significant increase in annexin II levels (bottom three panels of figure 5.3).

Although variations in annexin II levels between some of th e transfected clones appear to be greater th a n those observed for naturally occurring clonal variation in section 3.4 none were judged to be significant as cell loadings were approxim ate, and variations due to the transfected DNA were expected to be greater th a n those observed.

5.4 Riboprobe analysis of antisense transcripts

Since none of the transfected clones showed a reduction in annexin II protein expression levels, it was im portant to determine w hether antisense m essage was actually being transcribed. RNA was prepared from four of the pNV-XNA2-transfected clones as well as from the parent RBL cell line. A construct in pB luescript containing the first 900 bp of the annexin II cDNA, up to the Xho I site (see figure 5.4) w as used as

pNV XNA2 pNV XNA2 pNV XNA2 pNV XNA2 pNV XNA2 PNVANX2 pNVANX2 PNVANX2

F i g u r e 5 . 3 Western blots of RBL-2H3 clones transfected with constructs

containing annexin II cDNA in the sense and antisense orientations, immunoblotted for annexin II.

C l o n e s i n t h e t o p f i v e p a n e l s w e r e t r a n s f e c t e d w i t h p N V - X N A 2 a n d c l o n e s i n t h e b o t t o m t h r e e p a n e l s w e r e t r a n s f e c t e d w i t h p N V - A N X 2 . T h e l e f t - h a n d t r a c k i n e a c h p a n e l c o n t a i n s c e l l l y s a t e f r o m t h e p a r e n t R B L - 2 H 3 l i n e .

a tem plate for in vitro transcription of RNA. Radiolabelled sense stra n d RNA w as produced by small-scale in vitro transcription from the plasmid (linearised w ith Xho I) using th e T3 RNA polymerase promoter located a t the 5’ end of the insert. The plasm id w as linearised with Bam HI, and RNA w as transcribed with T7 RNA polym erase to produce unlabelled antisense annexin II RNA to be used as a positive control.

BamH I

4

Xho I

5’ AnxHcDNA 3

pBluescript

Figure 5.4 Schematic representation o f the template fo r in vitro transcription of sense and antisense annexin JT.

This figure shows the restriction sites used to linearise th e plasm id and the T7 and T3 RNA polymerase prom oters (empty boxes) located either side of the inserted 900bp of annexin n cDNA (filled box).

To determ ine the quantity of positive control RNA synthesised, th e incorporation of [^^P]-rCTP into trichloroacetic acid (TCA) insoluble m aterial w as determ ined for a portion of the reaction mixture and the am ount of RNA synthesised calculated a s described in Materials and Methods. 33% of the CTP was incorporated, th u s 21. Ipg of RNA were synthesised (2.1pg/|il). Various loadings of positive control RNA were

subjected to denaturing RNA gel electrophoresis alongside 20pg of RNA from each clone. The gel w as N orthern blotted and probed with radiolabelled sense stra n d RNA. The autoradiograph of th e riboprobe is shown in figure 5.5.

EXren th o ug h as little as Ing of the positive control RNA w as detected, th ere w as no signal in any of the RBL-2H3 cell tracks after overnight exposure to X-ray film. Exposure to a phosphorlmager plate for 18 hours (not shown) revealed a b a n d in the “lOOpg” track of positive control RNA b u t failed to detect anything in th e tra c k s from

R B L - 2 H 3 t r a n s f e c t a n t s . S i n c e l O O p g i s e q u i v a l e n t t o a p p r o x i m a t e l y 1 c o p y p e r c e l l ( D r .

P . S m i t h , p e r s o n a l c o m m u n i c a t i o n ) i t w a s a p p a r e n t t h a t l i t t l e o r n o t r a n s c r i p t i o n o f t h e

a n t i s e n s e o r i e n t a t i o n a n n e x i n II c D N A w a s o c c u r r i n g i n a n y o f t h e s e c l o n e s .

P o s i t i v e c o n t r o l R N A p N V X N A 2 c l o n e s

l O n g I n g l O O p g l O p g R B L 1 2 3 4

F i g u r e 5 . 5 Riboprobe blot of annexin 27 antisense transcripts in RBL-2H3

cells transfected with pNV-XNA2

T h e f i r s t f o u r t r a c k s c o n t a i n t h e i n d i c a t e d a m o u n t s o f in vitro t r a n s c r i b e d p o s i t i v e c o n t r o l R N A . T h e f i f t h t r a c k c o n t a i n s R N A i s o l a t e d f r o m p a r e n t R B L - 2 H 3 c e l l s . T h e r e m a i n i n g t r a c k s c o n t a i n R N A i s o l a t e d f r o m f o u r p N V - X N A 2 - t r a n s f e c t e d c l o n e s . T h e c o n t r o l t r a n s c r i p t s a r e a p p r o x i m a t e l y 9 0 0 b a s e s ; t h e a n t i s e n s e t r a n s c r i p t s i n R N A i s o l a t e d f r o m t r a n s f e c t e d c l o n e s s h o u l d b e l . S k b . F r o m t h i s i t w a s c o n c l u d e d t h a t a l t h o u g h t h e s i n g l e p r o m o t e r r e s p o n s i b l e f o r d r i v i n g t r a n s c r i p t i o n o f t h e n e o m y c i n r e s i s t a n c e g e n e a n d t h e i n s e r t e d c D N A w a s a c t i v e i n R B L - 2 H 3 c e l l s , i t d i d n o t a p p e a r t o p r o d u c e t r a n s c r i p t s f r o m t h e c D N A . T h i s c o u l d b e b e c a u s e t h e p r o m o t e r c a n a c t a t t h i s d i s t a n c e t o p r o m o t e R N A p o l y m e r a s e b i n d i n g t o t h e s t a r t o f t h e n e o m y c i n g e n e , b u t t h e p o l y m e r a s e d o e s n o t r e c o g n i s e t h e s t a r t o f t h e i n s e r t e d a n t i s e n s e D N A . A l t e r n a t i v e l y t h e p r o m o t e r m a y b e o n l y w e a k l y a c t i v e i n t h e s e c e l l s - a d e q u a t e f o r t h e c e l l s t o s u r v i v e i n n e o m y c i n b u t n o t e n o u g h t o g e n e r a t e d e t e c t a b l e q u a n t i t i e s o f a n t i s e n s e t r a n s c r i p t s . A n o t h e r p o s s i b l e e x p l a n a t i o n i s t h a t w h e n t h e p l a s m i d b e c o m e s i n c o r p o r a t e d i n t o t h e h o s t g e n o m i c D N A , i t p r e f e r e n t i a l l y l i n e a r i s e s a t a p o i n t b e t w e e n t h e p r o m o t e r a n d t h e s t a r t o f t h e i n s e r t o r i n t h e i n s e r t

Itself. In th is case cells will only survive If the neomycin resistance gene Is Inserted Into a region of genomic DNA th a t Is actively transcribed, close to a prom oter th a t stim ulates transcription from It. This would contribute to the low efficiency of stable transfection.

Based on these results, no further transfections were attem pted with plasm ids constructed In pNV and It was decided to consider remaking th e co n stru cts In a different vector, a plasm id called pRc/CMV (described In M aterials a n d Methods). This plasm id h a s the advantage of separate promoters for the neomycin resistance gene and the Inserted DNA.