Reporter genes for in vivo transient gene expression studies in tilapia (Oreochromis aureus) and common carp (Cyprinus carpio) one celled embryos

Theriogenology41:240,

1994

R E P O R T E R GENES FOR IN V I V O T R A N S I E N T GENE E X P R E S S I O N S T U D I E S IN T I L A P I A ( O r e o c h r o m i s aureus) A N D C O M M O N C A R P ( C y p r i n u s carpio) ONE C E L L E D E M B R Y O S R.Lleonart, R.Mart~nez, D . G a r c f a del Barco, O.Hern~ndez, F.O. Castro and J. de la F u e n t e M a m m a l i a n C e l l G e n e t i c s D i v i s i o n . C e n t r o de I n g e n i e r ~ a Gen~tica y Biotecnologfa. P.O. Box 6162, Havana, CUBA T r a n s i e n t gene e x p r e s s i o n assays, t o g e t h e r w i t h the use of r e p o r t e r genes, h a v e b e e n c r u c i a l to o b t a i n m o s t of the c u r r e n t k n o w l e d g e about gene r e g u l a t i o n in e u k a r y o t i c organisms. A l t h o u g h c u l t u r e d c e l l s are w i d e l y e m p l o y e d to s t u d y gene expression, results d e r i v e d from them are c o n s i d e r e d as in v i t r o o b t a i n e d data and o f t e n do not r e f l e c t p r o p e r l y h o w c e r t a i n genes are r e g u l a t e d in vivo. In this r e p o r t w e s h o w the feasibility of injecting chloramphenicol acetyl t r a n s f e r a s e (CAT) a n d h e p a t i t i s B s u r f a c e a n t i g e n (HBsAg) r e p o r t e r genes into single cell tilapia a n d / o r carp e m b r y o s as a w a y to test c o n s t r u c t s in c o n d i t i o n s m u c h c l o s e r to the l i v e o r g a n i s m . T w o p l a s m i d s w e r e u s e d in the e x p e r i m e n t s : p E 3 0 0 , w h i c h h a s the C A T g e n e u n d e r the c o n t r o l of h u m a n cytomegalovirus (CMV) e n h a n c e r - p r o m o t e r r e g i o n a n d 3' p o l y a d e n y l a t i o n s e q u e n c e s from the SV40 genome, and pUCHBV, w h i c h c o n t a i n s the e n t i r e h e p a t i t i s B v i r u s (HBV) g e n o m e e x c e p t for the c o r e s e q u e n c e , i n s e r t e d in p U C I 9 . P l a s m i d s w e r e d i l u t e d to 50 ~g/ml in i n j e c t i o n b u f f e r (i0 m M T r i s H C l p H 7.4, 0.25 m M EDTA, 0.1% Phenol Red) and about 1 nl was injected into one c e l l e m b r y o s , 30 to 60 m i n . a f t e r fertilization. I n j e c t e d embryos w e r e i n c u b a t e d for 2-4 days a n d b e f o r e h a t c h i n g w e r e t r a n s f e r r e d to e p p e n d o r f tubes, r e s u s p e n d e d in i00 ~I of 250 m M T r i s H C l p H 8, a n d a b o u t 50 ~i of g l a s s b e a d s ( 0 . 5 - 1 mm) w e r e a d d e d . Tubes were vortexed for 1 minute, f r e e z e - t h a w e d 3 times (-70°C,+37°C) r e m a i n i n g 5 m i n at each temperature, and after the a d d i t i o n of 1 ~i of 0.5 M EDTA, the tubes w e r e h e a t e d I0 m i n at 60°C. CAT a s s a y was done as s t a n d a r d procedures, and the H B s A g was d e t e r m i n e d b y E L I S A in e m b r y o h o m o g e n a t e s . T h e i n j e c t i o n p r o c e d u r e s h o w e d v a r i a b i l i t y in the v i a b i l i t y of e m b r y o s between experiments. V i a b i l i t y v a r i e d f r o m 1 0 % to 60%, d e p e n d i n g on the s k i l l of the o p e r a t o r , the q u a l i t y of the eggs t h e m s e l v e s , a n d the i n c i d e n c e of c o n t a m i n a t i o n d u r i n g incubation, among other factors. The results from several e x p e r i m e n t s s h o w e d a h i g h f r e q u e n c y of p o s i t i v e embryos (4050%), r e g a r d l e s s of the r e p o r t e r g e n e used. CAT e x p r e s s i o n r a n g e d b e t w e e n 5 to 50% c o n v e r s i o n of c h l o r a m p h e n i c o l to its a c e t y l a t e d forms, as j u d g e d b y c a r e f u l e x a m i n a t i o n of the a u t o r a d i o g r a p h i c films. E x p r e s s i o n of the H B s A g was o b s e r v e d in a p p r o x i m a t e l y 50% of the i n j e c t e d tilapia embryos w i t h a m e a n v a l u e of 0 . 7 4 + / - 0 . 1 6 n g / m l of h o m o g e n a t e . T h i s e a s y p r o c e d u r e m a y be u s e f u l w h e n s e v e r a l c o n s t r u c t s h a v e to be t e s t e d for t h e i r s t r e n g t h , e m p l o y i n g one of the g e n e s as a r e p o r t e r and the s e c o n d as an internal s t a n d a r d to n o r m a l i z e for i n t r i n s i c v a r i a t i o n s of the s y s t e m ( a m o u n t of p l a s m i d injected, e f f i c i e n c y of injection).

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Copyright © 1994 Butterworth-Heinemann