being cultured for seven days change the medium everyday then the clones were picked out 3 characterization of the embryonic stem cells knock in human ngf mature peptide mouse as shown in fig3 a the genomic dna of es cells and g418 fiau double resistant clone were extracted and digested with ecor i and then southern blotting was performed by using the probe a of 5 u2032 end targeting vector there was only one band about 10 kb in wild type cells compared with the recombinant es cells which show another band about 5 kb see fig3 b because of the ecor i site in human ngf mature peptide gene as shown in fig4 a primer 1 5 u2032 gctcatcctcccatcccatcttccaca 3 u2032 seq id no 13 locates at the 5 u2032 end of the mature peptide and primer 2 5 u2032 gaacgagatcagcagcctctgttc ca 3 u2032 seq id no 14 locates at neo gene when using primer 1 and primer 2 to amplify the genome there is not any band in wild type es cells but in recombinant es cells there is a band about 1200 bp amplified fig4 the pcr product was verified by ecor i digestion and dna sequencing and confirmed that the ngf mature peptide gene in mouse genome has already been replaced with that of human genome 1 the method to construct mutant ngf knock in mouse embryonic stem cells is the same as example 3 2 the characterization of mouse.