Escuelas que dan seguimiento a la filosofía del Feng Shui

T o g e n e ra te tra n s g e n ic m ic e , th e A F T l-L C R h C D 2 m in ig e n e ( w it h p r o k a r y o t i c s e q u e n c e s r e m o v e d ) w a s i n j e c t e d i n t o (C B A xC 5 7 B l/1 0 )x(C B A xC 5 7 B l/1 0 ) F2 m ouse em bryos and seven A F T l-L C R h C D 2 fo u n d e r transgenic m ice w ere generated. Presence o f the transgene w as d e te rm in e d b y s lo t-b lo t and expression o f the tra n sg en ic h C D 2 w as assessed b y flo w cyto m e try. B lood cells fro m transgenic fo u n d e r m ice w ere s ta in e d w it h flu o re s c e in (F IT C ) c o n ju g a te d M A b a g a in s t e n d o g e n o u s m C D S a , a p h y c o e r y th r in (PE) co n ju g a te d M A b a g a in s t m C D 4 , and b io tin y la te d M A b-206 against hC D 2 (w ith stre p ta vidin -R e d 7 6 0 used as the second layer) and then analysed b y flo w c y to m e try (also re fe rre d as FACS analysis), g a ting on lym ph o cyte s b y size. Expression o f the transgenic hC D 2 w as observed in a ll seven fo u n d e rs th a t w e re s u b se q u e n tly b re d to n o n tra n s g e n ic C B A m ice. Seven in d e p e n d e n t lin e s w e re o b ta in e d a n d the expression p a tte rn o f the A F T l-L C R hC D 2 transgene on the th ym o cyte s was analysed b y FACS as described above. The sites o f transgene in te g ra tio n w it h in the m ouse genom e w ere d e te rm in e d u s in g the Fluorescence In S itu H y b rid is a tio n (FISH) analysis (co lla b o ra tio n w it h D rs A . S aveliev, M . Fox and R. Festenstein).

A s it has been a lre a d y dem onstrated th a t d e le tio n o f the HSS3 re g io n fro m th e h C D 2 m in ig e n e re su lts in PEV, w e p re d ic te d th a t d e le tin g th e F T l sequences m a y also re su lt in the va rie g a tio n o f expression o f the A F T l-L C R h C D 2 transgene. A n a ly s is o f the transgene expression o n th y m o c y te s has

sh o w n th a t tw o transgenic lines, designated F T l. l and F T l.2, had n o tice a b ly m o re h C D 2 n egative cells as com pared w ith the n o n v a rie g a tin g M g 4 lin e used as a b a ckg ro u n d c o n tro l (the M g4 lin e carries the hC D 2 m in ig e n e w it h in ta c t LC R inte gra te d in the centrom ere). In a d d itio n , it w as fo u n d th a t the F T l.7 lin e contained litte rm ate s w ith tw o transgene expression phenotypes. O ne g ro u p o f the litte rm a te s (nam ed FT1.7B) had n o tic e a b ly m o re h C D 2 n e g a tiv e th ym o cyte s, w hereas the o th e r g ro u p (nam ed FT1.7A) h a d v e ry fe w h C D 2 n e g a tiv e th y m o c y te s , c o m p a ra b le w it h th a t in th e M g 4 b a c k g ro u n d c o n tro l line.

To q u a n tita te the degree o f va rie g a tio n , w e used a p a ram eter, d e signated KpEv and calculated as a percentage o f thym o cyte s fa llin g ou tside o f 1.3xSD (S ta n d a rd D e v ia tio n ) in te rv a l m easured fro m the m ean o f the p e a k o f h C D 2 p o s itiv e thym ocytes. It sh o uld be noted, th a t i f a peak is in a shape o f the n o rm a l d is trib u tio n , then Kpev is equal to 10%. Such ca lcu la tio n s (see m a te ria ls an d m ethods) fo r the M g 4 lin e re s u lte d in average Kp e v= 9 %, in d ic a tin g th a t th e tra n s g e n ic h C D 2 m in ig e n e expresses u n im o d a lly v ir tu a lly on a ll th ym o cyte s, so th a t the peak's shape is v e ry close to the n o rm a l d is tr ib u tio n (F ig ure 21,25). S im ila r c a lc u la tio n fo r the F T l . l lin e re s u lte d in average K p e v = 2 9 % , in d ic a tin g th a t in th is lin e the n u m b e r o f

v a rie g a tin g cells is 20% h ig h e r th a n in the n o n v a rie g a tin g M g 4 c o n tro l lin e . The F IS H e x p e rim e n ts have s h o w n th a t th e A F T l-L C R h C D 2 transgene in the F T l. l lin e w as inte gra te d in the centrom ere (F igure 21,25). The same ca lcu la tio n s fo r the F T l.2 and FT1.7B m ice re su lte d in average KpEv=16% and 28%, w hereas the FISH e xp erim e nts have s h o w n th a t the tra n s g e n e w as in te g ra te d at th e c e n tro m e re a n d te lo m e re b o rd e rs , re s p e c tiv e ly (F ig ure 22,25). In contrast, the M g 4 c o n tro l lin e c a rry in g the h C D 2 m in ig e n e w it h an in ta c t LC R in te g ra te d in the centrom ere d id n o t variegate (Festenstein et al., 1996).

The re m a in in g fo u r A F T l-L C R hC D 2 transgenic lines (n a m e ly FT l.3,4,5,6) expressed the transgenic hC D 2 in a fa ir ly u n im o d a l m anner, re s u ltin g in an average Kpev w ith in the 8-13% range. The FISH analysis has s h o w n th a t the A F T l-L C R hC D 2 transgene in these lines inte gra te d in the lo n g arm s o f the chrom osom es (F igure 23,24,25).

A s u m m a ry o f degree o f v a rie g a tio n fo r a ll seven tra n sg en ic A F T l-L C R h C D 2 lin e s is present on F ig u re 25. The d ia g ra m d em onstrates th a t the F T l. l lin e c a rry in g the transgene in te g ra te d in the c e n tro m e re has the h ig h e st KpEv- The F T l.2 and F T l.7 m ice carried the transgene in te g ra te d at the ce n tro m e re and te lo m ere b o rd e rs, re s p e c tiv e ly , and h a d a r e la tiv e ly h ig h n u m b e r o f CD2 negative thym ocytes, a lth o u g h n o t as h ig h as in F T l. l. T herefore, i t appears th a t the d e le tio n o f the F T l sequence fro m th e LC R renders the A F T l-L C R hC D 2 m in ig e n e subject to PEV w h e n the transgene is in te g ra te d in to the h e te roch ro m a tic regions (such as the ce n tro m ere o r telom ere) o r at th e ir borders.

F ig u re 25 show s th a t in n o n v a rie g a tin g lines the Kpev p a ra m e te r does n o t v a r y s ig n ific a n tly a m o n g litte rm a te s o f th e same lin e . T h u s , a ll 17 litte rm a te s o f the M g 4 lin e ha d Kpev iri the 8 to 10% range. S im ila rly , 6 litte rm a te s in the n o n v a rie g a tin g F T l. 3 a n d F T l. 4 lin e s h a d Kp e v co n sisten tly in the 7 to 9% range.

Q u ite a d iffe re n t p ic tu re was observed in the v a rie g a tin g lines. W e fo u n d th a t the litte rm a te s in the F T l.7 lin e , c a rry in g the transgene in te g ra te d at the te lo m ere b o rd e r, c o u ld be d iv id e d in to tw o g ro u p s a cco rd in g to th e ir transgene e xp ressio n p h e n o ty p e . A s the m a m m a lia n te lo m e re -in d u c e d PEV has n o t been described in d etail, it is n o t clear w h e th e r such an effect is a ttrib u te d o n ly to the te lo m e ric PEV. H o w e v e r, the Kpev p a ra m e te r was also v a rie d am ong the litte rm a te s o f the v a rie g a tin g F T l. l and F T l.2 lines (w ith ce n tro m ere -ind u ce d PEV), a lth o u g h n o t to such a d ra m a tic e xte n t as

in F T l. 7. For exam ple, o u t o f the 18 m ice analysed fo r the F T l. l lin e , one m ouse had Kpev= 13%, another 38% and the re m a in in g m a jo rity h a d Kpev close to 28% (re su ltin g in the average o f 29%).

To exclude the p o s s ib ility o f d iffe re n t transgene in te g ra tio n s as a reason fo r the diffe re nce s in v a rie g a tio n p h e n o typ e s a m o n g the FT1.7A and FT1.7B litte rm a te s . S ou th e rn B lo t analysis w as p e rfo rm on ge no m ic D N A fro m these m ice. In a d d itio n , tw o F T l. l litte rm a te s w it h h ig h e st and the lo w e s t K p e v w e re also analysed b y S outhern B lo t (K a th le e n R o d e rick, data n o t show n). The e xp e rim e n t has dem on stra ted th a t the v a ria tio n in the degree o f PEV w it h in one lin e is n o t caused b y d iffe re n t in te g ra tio n s and th a t the transgene in the F T l. l and F T l.7 v a rie g a tin g lin e s are n o t rearranged.

The exact reason fo r such a v a ria tio n in degree o f PEV is n o t clear b u t it is p o s s ib le th a t th e degree o f v a rie g a tio n is in flu e n c e d b y the g e n e tic b a c k g ro u n d (Festenstein and K ioussis, pe rson a l c o m m u n ica tio n ).