Prolonged culturing of avian (chicken) primordial germ cells (PGCs) using specific growth factors, use thereof to produce chimeric avians (chickens)

Abstract

A culture system for maintaining avian PGCs for long periods in tissue culture is provided. This culture system uses LIF, bFGF, IGF and SCF. The resultant PGCs are useful for the production of transgenic and chimeric avians, in particular, chickens or turkeys.

1 . A culturing method which provides for maintenance of avian primordial germ cells for prolonged periods comprising the following steps: 
 (i) isolating primordial germ cells from a desired avian; and    (ii) culturing said primordial germ cells in a culture medium containing at least the following growth factors contained in amounts sufficient to maintain said PGCs for prolonged periods in tissue culture: 
 (1) leukemia inhibitory factor (LIF),  
 (2) basic fibroblast growth factor (bFGF),  
 (3) stem cell factor (SCF) and  
 (4) insulin-like growth factor (IGF), for a prolonged time period.  
   
     
     
         2 . The method of  claim 1 , wherein the minimal amounts of said growth factors are: 
 (1) LIF (0.00625 U/μl,    (2) bFGF (0.25 pg/μl),    (3) IGF (0.5625 pg/μl), and    (4) SCF (4.0 pg/μl).    
     
     
         3 . The method of  claim 2 , wherein the maximal amounts of said growth factors range from about two times to one hundred times said minimum amounts.  
     
     
         4 . The method of  claim 1 , wherein said avian PGCs are obtained from an avian of the genus  Gallinacea.    
     
     
         5 . The method of  claim 4 , wherein said PGCs are chicken PGCs or turkey PGCs.  
     
     
         6 . The method of  claim 1 , wherein said PGCs are maintained in culture for at least 14 days.  
     
     
         7 . The method according to  claim 6 , wherein said PGCs are maintained in culture for at least 25 days.  
     
     
         8 . The method according to  claim 7 , wherein said PGCs are maintained in culture for at least 4 months.  
     
     
         9 . The method of  claim 1 , which further comprises: 
 (iii) transfecting or transforming the resultant PGCs with a desired nucleic acid sequence.    
     
     
         10 . The method of  claim 9 , wherein said nucleic acid sequence encodes a therapeutic polypeptide.  
     
     
         11 . An improved method of producing chimeric avians which comprises: 
 (i) isolating primordial germ cells from an avian;    (ii) maintaining such PGCs in a tissue culture medium containing at least the following growth factors; 
 (1) leukemia inhibitory factor (LIF),  
 (2) basic fibroblast growth factor (bFGF),  
 (3) stem cell factor (SCF) and  
 (4) insulin-like growth factor (IGF);  
   (iii) transferring said PGCs into a recipient avian embryo; and    (iv) selecting for chimeric avians which have the desired PGC phenotype.    
     
     
         12 . The method according to  claim 11 , wherein said PGCs are derived from avian embryos of the genus  Gallinacea.    
     
     
         13 . The method according to  claim 12 , wherein said avian embryos are turkey or chicken embryos.  
     
     
         14 . The method according to  claim 11 , wherein said PGCs are transfected or transformed with a desired nucleic acid sequence prior to transferral to a recipient avian embryo.  
     
     
         15 . The method according to  claim 14 , wherein said nucleic acid sequence encodes a therapeutic polypeptide.  
     
     
         16 . The method according to  claim 15 , which further includes purifying said therapeutic polypeptide from the eggs of the chimeric avians produced according to step (iv).  
     
     
         17 . The method according to  claim 11 , wherein the PGCs are injected into the dorsal aorta and/or marginal vein of a recipient avian embryo or into recipient blastoderms.  
     
     
         18 . An avian PGC cell line obtained by the culturing method of  claim 1 .  
     
     
         19 . The cell line of  claim 18 , which is a chicken or turkey PGC cell line.  
     
     
         20 . The cell line of  claim 18 , which contains an inserted nucleic acid sequence.