Fluorescence A c tiv a te d C ell S orter
F o r the e v a lu a tio n o f the p a tte rn an d le v e l o f h C D 2 e xp ressio n , 10^ th ym o c y te s , m esenteric ly m p h node cells o r p e rip h e ra l b lo o d cells w e re in cu b a te d fo r 30 m in u te s at 4 °C w it h C D4 re d (B o e h rin g e r M a n n h e im ), CD8 PE (Catlag Laboratories) and FITC conjugated o r b io tin y la te d a n ti-hC D 2 ( O K T ll (V e rb i et al., 1982) a ntibodies. Lysis o f red cells w as done u s in g B e cto n D ic k in s o n L y s is S o lu tio n a c c o rd in g to th e m a n u fa c tu re rs in s tru c tio n s . C ells w ere analysed u s in g a Beckton D ic k in s o n FACS sorter. T h re e c o lo u r a n a ly s is w as d o n e u s in g th e L y s is I I an d C e llQ u e s t p rogram m es (H e w le tt Packard and Becton D ickin so n , respectively).
Sam ples o f 10^ cells w ere tra n sfe rre d to 3 m l o f m e d iu m in Falcon tubes and c e n trifu g e d at 1,200 rp m fo r 7 m in . C ell pellets w ere resuspended in 50 m l s ta in in g s o lu tio n co n ta in in g antibodies (-1 0 m g / m l each) in PBS w it h 1 % BSA and 0.02 % azide. In the case o f the b lo o d cell analysis, red cells w ere lysed b y a d d in g 2 m l o f the Gey's lysis b u ffe r and in c u b a tin g at 37°C fo r 3 m in . C ells w ere stained w ith a second la ye r o f an tibo d ies in the same w a y , and w ere analysed b y FACScan (Becton D ickinson).
C a lc u la tio n o f Kpev
KpEv w as calculated as a percentage o f th ym ocytes fa llin g o u tsid e 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 th y m o c y te s . I f a p e a k is in a shape o f th e n o rm a l
d is trib u tio n , th e n Kpev is equal to 10%. Such calcu la tion s fo r the M g 4 lin e resulted in average K p e v = 9 % , in d ic a tin g th a t the transgenic hC D 2 m in ig e n e
expresses u n im o d a lly v ir tu a lly on a ll thym o cyte s, so th a t th e peak's shape is v e ry close to the n o rm a l d is trib u tio n .
KpEv o f the v a rie g a tin g lines was calculated as a percentage o f th ym o cyte s fa llin g o u tsid e o f l.S xS D in te rv a l m easured fro m the m ean o f the peak o f hC D 2 p o s itiv e thym ocytes u sin g S tandard D e v ia tio n o f the n o n v a rie g a tin g c o n tro l M g 4 lin e (SDM g4)- N.B.: fo r the lines w it h the transgene expression le v e l s ig n ific a n tly d iffe re n t fro m th a t o f M g4, the used SD was a d ju ste d according to the fo rm u la SD=SDM g4 x P /P M g 4 , w he re P and PM g4 are the means o f the peaks o f hC D 2 p o s itiv e thym ocytes in the considered lin e and M g 4 c o n tro l respectively.
G enom ic D N A E x tra c tio n
G enom ic D N A fro m m ouse ta ils was extracted as described in (H ogan et al., 1986). B rie fly , ta il tissues (about 5 m m ) w ere incubated o v e rn ig h t in 500 p i o f ta il m ix w it h 500 p g / m l p ro te in a se K (B o e h rin g e r M a n n h e im ) in a shaking w a te r b a th at 55®C. Samples w ere treated fo r 1 h o u r w it h 10 m g / m l R N A se A (Sigm a) at 3 7 °C and th e n w e re e xtracted tw ic e w it h an e q u a l a m o u n t o f p h e n o l/ c h lo ro fo rm and once w it h c h lo ro fo rm . S u b seq u e n tly the D N A w as p re c ip ita te d w it h 0.6 v o lu m e o f is o -p ro p a n o l fo llo w e d b y c e n trifu g a tio n at 13,000 rp m in a bench to p c e n trifu g e . The D N A w as w ashed in 70% ethanol, a ir d rie d b r ie fly and th e n resuspended in d d H 2 0 o v e r n ig h t a t 4 ° C. D N A c o n c e n t r a tio n w a s d e te r m in e d b y s p e c tro p h o to m e try .
D N A Probe Labelling
D N A pro be s w e re ra d io a c tiv e ly la b e lle d b y ra n d o m p r im in g u s in g a R andom P rim in g K it (B oehringer M a n n h e im ). 50 n g o f D N A resuspended in d d H 2 0 was denatured at lOO^C fo r 3 m inu tes, cooled im m e d ia te ly on ice fo r 5 m in u te s and in c u b a te d in a fin a l v o lu m e o f 50 p i w it h 10 p i o f R andom P rim in g B uffer, 5 p i o f P ^^-a d C T P (370 M B q /m l, A m e rsh am ) and 1 p i o f la b e llin g g ra d e K le n o w e n z y m e a t 3 7 ^ C fo r 30 m in u te s . U n in c o rp o ra te d n u cle o tid e s w ere re m o ve d u s in g a P harm acia G-50 n ic k c o lu m n a cco rd in g to the m a n u fa ctu re rs in s tru c tio n s . The collected p ro be was denatured b y b o ilin g at lOO^C fo r 5 m inu tes before use in h y b rid is a tio n reactions.
S lo t B lo t
10 m g o f ge no m ic D N A w as d e n a tu re d in 200 p i o f 4 M N a O H fo r 5 m in u te s at RT. A Schleicher and S chuell s lo t b lo t a p pa ra tu s w as set u p a cco rdin g to the m anufactu rers in s tru c tio n s u s in g a Schleicher and Schuell n itro c e llu lo s e m em brane w h ic h had been p re -w e tte d in 1 M a m m o n iu m acetate. The a p p a ra tu s w as th e n connected to a w a te r v a c u u m . D N A sam ples w e re n e u tra lis e d b y the a d d itio n o f 200 p i o f ice c o ld 2 M a m m o n iu m acetate and lo a d e d in d u p lic a te u s in g 200 p i o f the D N A sam ple p e r slot. A fte r lo a d in g , the filte r w as re m o v e d fro m the apparatus and the D N A was fix e d onto the filte r b y b a k in g fo r 2 h o urs at SO^C. Slot B lo t experim ents w ere carried o u t w ith the assistance o f K athleen R oderick.
Southern B lo t
10 jig o f genom ic D N A was digested in a fin a l v o lu m e o f 50 j i l o v e rn ig h t in the a p p ro p ria te c o n d itio n s fo r the re s tric tio n e nzym e u s in g 40 u n its o f enzym e. To check the c o m p le tio n o f d ig e s tio n , 3 p i o f th is re a ctio n w as m ix e d w it h 0.5 p g o f la m b d a D N A in a second e p p e n d o rf tu b e and in cu b a te d in p a ra lle l. The n e xt m o rn in g the la m b d a D N A sam ples w e re ru n on a 0.8% agarose gel to dete rm in e the extent o f digestion. A n y sam ple th a t had n o t been digested to c o m p le tio n as ju d g e d b y the la m b d a D N A b a nd p a tte rn w as digested fu rth e r d u rin g the d a y b y the a d d itio n o f an a d d itio n a l 40 u n its o f fresh re s tric tio n enzym e, re p e a tin g the la m b d a test digest, and was then checked again u s in g the lam bda D N A sample. D igests w ere th e n electrophoresed s lo w ly (2 V /c m ^ ) o v e rn ig h t in a 0.8% agarose gel fo llo w e d b y p h o to g ra p h y u n d e r sh o rt w ave u.v. lig h t. The gel was then p re p a re d fo r b lo ttin g b y s u b m e rg in g the gel fo r 60 m in u te s in B lo t I s o lu tio n . The gel w as th e n in c u b a te d fo r a fu r th e r 60 m in u te s b y im m e rs io n in to B lo t I I s o lu tio n . The gel w as th e n set u p fo r o v e rn ig h t b lo ttin g according to (Southern, 1975) and subsequently, the D N A w as fixe d onto the m em brane b y b a k in g at 80°C fo r 2 hours.
For p la s m id blots 1 p g o f D N A was digested in a fin a l v o lu m e o f 20 p i o f I x r e s tr ic tio n b u ffe r u s in g fiv e u n its o f re s tr ic tio n e n z y m e u n d e r th e a p p ro p ria te co n ditio n s.
F ilte r Hybridisation
F ilte r m em branes w ere p re -h y b rid is e d at 65^C fo r at least 2 h o urs in 20 m l o f h y b rid is a tio n b u ffe r in either H y b a id bottles placed in a ro ta tin g H y b a id oven o r in perspex boxes subm erged in a sha kin g w a te r bath. 20 m l o f fresh h y b rid is a tio n b u ffe r, in to w h ic h 50 n g o f labelled probe had been added was th e n used fo r h y b rid is a tio n o v e rn ig h t at 65®C. F o llo w in g h y b rid is a tio n , n itro c e llu lo s e filte rs w ere w ashed to a fin a l s trin g e n c y o f O.SxSSC, 0.1% ( w / v ) SDS at 65®C ch a n g in g w a s h in g s o lu tio n s a fte r 15 m in u te s each. N y lo n filte rs w ere w ashed in 40 m M S o d iu m Phosphate, 0.1% ( w / v ) SDS at 65®C in the same m anner. W a sh in g was c a rrie d o u t in p la s tic boxes in a shaking w a te r bath. Filters w ere then prepared fo r a u to ra d io to g ra p h y .
A u to ra d io g ra p h y
W ashed n itro c e llu lo s e o r n y lo n m e m bra n e s fr o m h y b r id is a tio n w e re w ra p p e d in Saran W ra p and exposed to K o d a k X -o m a t-A R o r F u ji N if-R X p h o to g ra p h ic film at -70®C usin g in te n s ify in g screens.
M em brane S trip p in g
R a d io a c tiv e p ro b e w as re m o v e d fro m h y b r id is a tio n m e m b ra n e s b y im m e rs io n o f the m em brane in to a b o ilin g s o lu tio n o f O.lxSSC, 0.1% SDS and le ttin g the s o lu tio n cool to RT. C o m p le te re m o v a l o f p ro b e w as co n firm e d b y exposure o f the m em brane to p h o to g ra p h ic film .
Transgene Copy Num ber D eterm ination
D N A slot blo ts w ere h y b rid is e d w ith labelled c D N A (hC D 2 and m C D2). The b lo ts w e re exposed on a P h o s p h o rlm a g e r screen and scanned. N u m e ric a l v a lu e s w e re o b ta in e d fo r each sa m p le u s in g Im a g e Q u a n t so ftw a re (M o le c u la r D ynam ics). The co p y n u m b e r w as calcu la te d fo r each s a m p le b y d iv id in g th e h u m a n s ig n a l b y th e m o u s e s ig n a l a n d sta n d a rd isin g to a transgenic o f k n o w n copy n u m b e r (CD 2.3; Greaves et al., 1989).
Fluorescence in S itu H y b rid is a tio n (FISH)
FISH analysis was p e rfo rm e d b y D rs A . Saveliev and R. Festenstein. B rie fly ,