34results about How to "Achieve knockout" patented technology

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

The invention provides an establishing method of a recoverable immortalized hepatic cell line. The establishing method comprises the following steps: separating a hepatic progenitor cell from liver in a mouse which is 12.5-14.5 days old in an embryonic period; guiding a retrovirus containing genes SV40T and HSV-TK into the hepatic progenitor cell; screening a monoclonal cell strain having a hepatic progenitor cell marker and having a function of differing to a mature hepatic cell; then guiding a retrovirus containing CD gene into the cell strain so as to obtain recoverable immortalized hepatic cell carrying double suicide genes. The cell strain can multiply in vitro to obtain the phenotype and functions of a normal hepatic cell, the safety of the immortalized cell becomes adjustable through controllable modes such as locus recombination and drug screening; when the cell is used in a human body, the biological safety of the immortalized cell is ensured to the greatest extent and the dangerousness of the immortalized cell is reduced; therefore, a reliable, safe and ideal hepatic cell material is provided for bioartificial liver technology.

The invention discloses a method for increasing the ovalbumin expression of Bacillus subtilis, and belongs to the field of genetic engineering. The invention improves the ovalbumin expression of Bacillus subtilis by increasing the specific growth rate of Bacillus subtilis. In the present invention, the wild-type 168 is used as the starting strain, and a plurality of recombinant strains with increased specific growth rate can be obtained by using the method of knockout of growth-related genes, adaptive evolution (Adaptive Laboratory Evolution, ALE) and the combination of the two. The specific growth rate of Bacillus to increase the expression of Bacillus subtilis ovalbumin provides a new idea and method for increasing the expression of Bacillus subtilis ovalbumin.

The invention belongs to the field of biotechnology and provides a systemic Plin1 gene knockout animal model construction and identification method thereof. The steps of the construction method are: designing sgRNA sequences according to the sequence before and after exon 2 of the Plin1 gene, constructing a Plin1 gene knockout vector, and performing in vitro transcription to obtain sgRNA; mixing sgRNA and Cas9 protein and then microinjecting it into mouse fertilized eggs to obtain whole body Sexual Plin1 knockout mice. The beneficial effect of this project is that a pair of sgRNA sequences with the highest scores were screened out, which ensured the success rate of the project; a large fragment of exon 2 of the Plin1 gene was knocked out, and the knockout efficiency was improved; the knockout region was designed 3 The two primers can successfully identify various genotypes of mice, avoiding the situation that two primers may not be able to distinguish genotypes when large fragments are knocked out. It provides a convenient, reliable and economical animal model and a good foundation for studying the biological function of PLIN1 and the pathogenesis of obesity-related metabolic diseases.