A Preliminary Study On The Technical System Of Aromatic Enzyme Gene Modified Chicken

Due to the special reproductive structure of the chickens,the hens produced the eggs in the body after fertilization and the chicken embryos in the eggs had developed into thousands of cells.In the process of production of transgenic chickens,primordial germ cells migration and the lentiviral vector transgenic method were used to produce chimeric chicken and with the next generation of breeding in order to obtain transgenic chicken,its efficiency and research time were questionable.Although there had aromatase gene expression regulation studies,so far there was no aromatase gene knockouted chicken.Therefore,my attempts were to establish a chicken embryos on technology systems.We used aromatase gene knockout vector to produce the aromatase gene knockout chicken and establish the gene modified chicken technology system for poultry Genomics research.We wanted to lay the foundation for poultry disease-related gene modification.In order to use chicken embryo to achieve the production of aromatase gene knockout chicken,this paper designed three aspects of research:We designed and validated the aromatase gene knockout vector.We designed six sg RNAs of aromatase gene knockout and constructed by the CRISPR / Cas9 technique.Sg RNA2 and sg RN A6 confirmed on chicken embryo fibroblasts could complet the gene knockout work.We established a vector system for the successful manufacture of knockout chickens.Then the constructed vector was ligated to the plasmid,and the chicken embryos by transient transfection were digested by T7E1.The results of the digestion showed that sg RNA2 and sg RNA5 combination contributed to the best results.Further target sites were found knockouted sections and sg RNA5 combinated with sg RNA2 worked good.We found no off-target in the five sites.Chicken embryos were researched on the method.As the chicken embryo position in the egg was uncertainty,We studied the chicken embryos placed 6 hours and 24 hours,we found chicken embryo deviation rate of 24 hours was minimum and the paraffin shell membrane Sealing method was better.The weight loss rate of eggs with different openings was not significant.Wiht the chicken embryo toxicity observed,the results showed that the chicken embryo development rate and survival rate were higher after injection of knockout plasmid.The production rate gradually decreased after added one and more affecting factors.The eggs were not produced by filling the eggs.Exploitation of Aromatic Enzyme Gene was made in Chicken.First,we used lentiviral vector GFP transfected chicken embryos and go t the development of transgenic chicken embryos for 3 days.O n the other hand,we transfected the chicken embryos with GFP plasmid and GFP(RNA)transfection and obtained green fluorescent in chicken embryos.After the DN A was extracted,we obtained the target bands after the PCR reaction.At the end of this experiment,no aromatase gene knockout chickens were made.Although the study did not produce genetically modified chickens,we used the non-viral method to make gene-modified chickens and the study of gender reversal was explored.Due to the special physiological characteristics of chickens,knockouted fragments was larger,affecting the development of chicken embryos and more factors,the experiment increased the difficulty.