H. E. LEHMAN 211 



when properly executed, contains the egg nucleus. In the hands 

 of Briggs and King, enucleation by this means was approximately 

 99% successful. Diploid donor cells were isolated from normal 

 blastulae, gastrulae, neurulae, and tail-bud embryos by microdis- 

 section, or better, by the trypsin-versene method for dissociating 

 embryonic cells ( King and Briggs, 1955 ) . Single cells were drawn 

 into fine micropipets in such a manner as to cause the cell mem- 

 brane to rupture without diluting the cell contents with the op- 

 erating medium. With the aid of an Emerson micromanipulator, 

 the whole cell was then injected into an activated and enucleated 

 host egg. No attempt was made to exclude the donor cytoplasm 

 which, however, constituted only a very minute fraction (1:20,- 

 000 to 1:600,000) of the total egg volume. It is significant in con- 

 nection with the results reported that differentiated cytoplasm 

 lacking nucleus never stimulated any development when injected 

 into enucleated host eggs ( Briggs and King, 1953 ) . 



The transplantation technique was first tested to determine 

 whether or not the various manipulations damaged either the 

 egg cytoplasm or the transplanted nucleus. Initial experiments 

 (Briggs and King, 1952) employed only undetermined prospec- 

 tive ectodermal cells of normal diploid blastulae. Approximately 

 one-third of the cases gastrulated and, of these, nearly half formed 

 embryos which were quite normal in appearance and capable of 

 developing into swimming larvae, and the remainder underwent 

 somewhat abnormal postgastrula development. Determinations 

 of ploidy in these embryos, based on nuclear size and nucleolar 

 number, revealed that all were either diploid or polyploid (hap- 

 loids would generally be expected if these cases represented 

 failures in enucleation of the egg followed by parthenogenetic de- 

 velopment; however, spontaneous regulation to diploidy can 

 occur in parthenogenetic frog eggs as Parmenter, 1952, and 

 others have shown ) . Although the percentage of successful cases 

 was somewhat lower, essentially similar results were obtained 

 when undetermined prospective neuro-epidermal cells of early 

 gastrulae were used as a source of donor nuclei ( Briggs and King, 

 1953; King and Briggs, 1954a, 1955). It thus seems certain that 

 these diploid nuclei from blastulae and early gastrulae were not 



