| Gene editing as a method of introducing new genetic elements into organisms has been around since the 1970's.While plenty of gene editing approaches are available for the gene function study,such as classical forward genetics techniques,which include UV mutagenesis,chemical mutagenesis,retrovirus and transposon-mediated insertion mutation,and the reverse genetics techniques that consist of TALEN,ZFN,and CRISPR/Cas9,the saturation mutagenesis of all endogenous genes within the mouse genome is not well understood.The Sleeping Beauty(SB)transposon system,which is composed of a SB transposase and a transposon that was designed in 1997 to insert specific sequences of DNA into genomes of vertebrate animals,has been used for introducing new traits and to discover new genes and their functions frequently.In this research,the SB transposon system was combined with a poly(A)-trap vector to study the insertional mutation of mouse embryonic stem cells(mES).This poly(A)-trap vector contained an expression cassette of neomycin(Neo)-resistant gene lacking a poly(A)signal and flanked by two inverted terminal repeats of the Sleeping Beauty(SB)transposon.The whole poly(A)-trap cassette was transposed into target TA dinucleotides,properly splice with endogenous genes and effectively interrupt the transcription of trapped genes in mES cells after transient induction of SB expression by doxycycline(DOX)-treatment at 1?g/ml,leading to the formation of multiple geneticin(G418)-resistant cell clones.In this study,two new poly(A)-trap vectors pT2/Poly(A)-trap-Short and pT2/Poly(A)-trap-ES,and one induced-transposase vector pCMV-rtTAˇTRE-SB11 which induced expression of SB 11 transposase were constructed firstly.The two poly(A)-trap vectors included a gene-identification cassette and a gene-breaking cassette.The transposase vector pCMV-rtTAˇTRE-SB11 induced the expression of SB 11 transposase by the Tet-on system,thereby increased the efficiency of mutation.The appropriate concentration and using time of doxycycline(DOX)is determined by the effects of DOX required by the Tet-on system on different levels of growth and proliferation of feeder cells and mouse embryonic stem cells.Then the obtained mutant mouse embryonic stem cell lines were tested for embryonic stem cell characteristics such as pluripotency,self-renewal ability,genetic stability,differentiation potential,etc.Also,the induction method and the screening method were identified through the transposon and capture vector.The results showed that the characteristics of mouse embryonic stem cells were not significantly damaged,and they were maintained as much as possible.At the same time,the scheme for constructing mutant mouse embryonic stem cell lines was optimized to some certain extent,and the transposition efficiency was increased from about 10%to about 42%.The complete methodology for the construction of mutant mouse embryonic stem cell lines based on the SB transposon was constructed through the aforementioned experimental research.After several times of mutation screening,six transposition events from twenty-three cell clones were identified.The abilities of self-renewal,totipotency,genetic stability and differentiation of syngapl+/-cells were not affected by DOX-treatment and G418-selection.These findings suggest that this SB transposon-mediated poly(A)-trap vector can be used as an alternative tool and a promising technique for the exploitation of the large-scale screening of mES cells with a gene mutation and for further generation of mutant mouse strains. |
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