Rearranged zebrafish genomic DNA induces zebrafish mutant after microinjection into fertilized egg and preliminary study of the mechanism

Genomic DNA of zebrafish was first digested incompletely with Msp I, and then the fragments were joined to form rearranged genomic DNA. This rearranged genomic DNA was incompletely digested with EcoR I, and the fragments were linked with a long adaptor. Two primers (Gmprimer1 and Gmprimer2) were designed according to the adaptor sequence for two-step amplification. The Gmprimer1-amplified products were microinjected into fertilized zebrafish eggs after purification and a red flesh mutant was observed among 42 surviving zebrafish. We obtained several introduced sequences by two-step amplification. The second set of Gmprimer2-amplified products were purified and microinjected into fertilized zebrafish eggs; all 37 surviving fish were red flesh mutants. We found that the largest amplified band from the mutant from the first microinjection was also present in the amplified pattern from six mutants from the second microinjection. The length of the sequence was 2,565 bp, but it did not encode any proteins. Microinjecting this sequence into fertilized zebrafish eggs produced the red flesh mutant. The sequences differed slightly among different individuals from the second microinjection. Most regions of these sequences were the same, w...Continue Reading