Many diseases, such as genetic disorders and tumors, have a close causal relationship with genetic abnormalities in the human body. The idea of introducing normal gene sequences into patients for gene level therapy has been proposed even before DNA recombination technology. Edward Tatum and Joshua Lederberg proposed the use of viruses as gene transfer vectors in the early 1960s.

The clinical trials of human cell gene therapy have begun. The following conditions must be met for gene therapy:

① Select appropriate diseases and have a clear understanding of their pathogenesis and the structure and function of corresponding genes;

② The gene for correcting the disease has been cloned, and the mechanism and conditions of gene expression and regulation are understood;

③ This gene has suitable receptor cells and can be effectively expressed in vitro;

④ Having safe and effective transfer carriers and methods, as well as available animal models.

In the past three years, clinical gene therapy has been carried out for several human monogenic genetic diseases and tumors.

The efficiency of correctly connecting the target sequence with the vector DNA and the efficiency of recombinant introduction into cells are not 100%, so the cells that grow and reproduce in the end do not all carry the target sequence. Generally, a carrier only carries a certain segment of exogenous DNA, and a cell only accepts one recombinant DNA molecule. Only a portion, or even a very small portion, of the cultured cell population is a recombinant containing the target sequence. Screening out the target recombinant is equivalent to obtaining a clone of the target sequence, so dcreening is an important step in gene cloning. When constructing vectors, selecting host cells, and designing molecular cloning schemes, careful consideration must be given to screening issues.

After connecting the target gene sequence with the vector, it needs to be introduced into cells for reproduction and amplification. After screening, recombinant DNA molecular clones can be obtained. Different vectors reproduce in different host cells, and the method of introducing into cells is also different.

At present, researchers use the method of introducing siRNA molecules into cells for RNAi research. There are currently 5 methods and pathways for synthesizing siRNA molecules:

1. Chemical synthesis;

2. In vitro transcription;

3. "Cocktail" method (RNaseIII enzyme digestion of dsRNA);

4. In vivo expression of siRNA mediated by plasmids and viral vectors;

5. siRNA expression box mediated siRNA in vivo expression.

How to design effective siRNA has always been a hot topic in current RNAi research The effectiveness of gene suppression largely depends on the selection of the target gene sequence. The target sequence can be randomly selected or tested on different regions of the target gene to determine which sequence is most effective. The following points are extremely important for the success of siRNA design.