| 摘要 |
Suggested is a method for enhancing the expression of taste related receptor genes encompassing the following steps:
(i) providing a culture of mammalian cells, the genome of said cells comprising at least one sweet receptor domain;
(ii) designing at least one type of single-guide RNA (sgRNA), the 10 to 30 nt guide sequence of said sgRNA being complementary to stretches within the non-coding and/or putative regulatory region upstream of the translation start codon of at least one sweet receptor gene;
(iii) preparing a vector comprising an expression cassette encompassing at least one optionally modified CRISPR-Cas9, preferably CRISPR-dCas9VP64, and at least one optionally modified sg-RNA optionally containing aptamer structures for binding activator proteins;
(iv) transfecting said culture of mammalian cells with said vector to target the genome for the presence of a DNA sequence that is complementary to the 10 to 30 nt guide sequence of said sgRNA; and
(v) measuring the transcriptional enhancement of the sweet receptor mRNA by quantitative RT-PCR. |
