The effective material basis of traditional Chinese medicine(TCM) is an indispensable part of studies on TCM, and each technology has its advantages and disadvantages. The target constituent knock-out/knock-in technology has attracted much attention since it was proposed because of its unique advantages of regarding the extract of the formula as a whole, which can better reflect the characteristics of multi-component and multi-target integration and regulation of TCM. This method investigated the contribution of target constituent to the overall efficacy of a TCM by analyzing the changes in efficacy of the remaining formula before and after knock-out/knock-in of the target constitution. The application of this model not only facilitates studies of the effective constituents of TCM, but also help to develop the quality control standard of TCM. However, the application of this model is restricted due to the limitation of target constituent separation technology. By reviewing the literatures in recent years, this study summarized the research process and application of this method for a reference.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Medicine, Chinese Traditional ; Quality Control

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.
Gene Expression Regulation, Fungal ; Gene Knock-In Techniques ; Genes, Reporter ; Genome, Fungal ; Saccharomyces cerevisiae ; genetics

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsI inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.
Biological Transport ; Escherichia coli ; enzymology ; genetics ; Gene Deletion ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Glucose ; metabolism ; Phosphoenolpyruvate Sugar Phosphotransferase System ; genetics ; Zymomonas ; genetics

The study aims to use CRISPR/Cas9 introducing foreign gene targeted knock-in into chicken EAV-HP genome. First, specific primers were designed for amplification of EAV-HP left, right homologous arms and enhanced green fluorescent protein (eGFP) expression cassette. PCR products of homologous arms were ligated to both sides of eGFP by overlap extension PCR, resulting in full-length donor DNA fragment designated as LER. Then LER fragments were cloned into pMD19-T to obtain donor vector pMDT-LER. Subsequently, the donor vector pMDT-LER was transfected into HEK293T cells to verify the expression of eGFP gene. Furthermore, co-transfection of CRISPR/Cas9 expression vector and pMDT-LER into chicken DF-1 cells was performed to achieve eGFP transgenic cells. Meanwhile, eGFP expression was observed in cells, and the event of eGFP integration into EAV-HP genome was detectable by amplification of target DNA. Finally, the transgenic DF-1 cells were passaged seven times, and the stable integration and expression of eGFP was checked by PCR and Western blotting. These results demonstrated that eGFP gene was knocked into the EAV-HP genome successfully, which provides a new integration site for research of transgenic chicken.
Animals ; CRISPR-Cas Systems ; Chickens ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knock-In Techniques ; Genome ; HEK293 Cells ; Humans

This study aimed to explore the expression changes of VASA gene in sheep testis development and to construct VASA gene knock-in vector to prepare for the study on the differentiation of sheep germ cells in vitro. The testicular tissues of 3-month-old (3M) and 9-month-old (9M) sheep which represent immature and mature stages, respectively, were collected. The differential expression of VASA gene was analyzed by quantitative real-time PCR (qPCR) and Western blotting, and the location of VASA gene was detected by immunohistochemistry. The sgRNA targeting the VASA gene was designed and homologous recombination vectors were constructed by PCR. Subsequently, plasmids were transferred into sheep ear fibroblasts. The VASA gene was activated in combination with CRISPR/dCas9 technology to further verify the efficiency of the vector. The results showed that the expression level of VASA gene increased significantly with the development of sheep testis (P < 0.01), and was mainly located in spermatocytes and round spermatids. The knock-in vector of VASA gene was constructed by CRISPR/Cas9 system, and the Cas9-gRNA vector and pEGFP-PGK puro-VASA vector were transfected into ear fibroblasts. After CRISPR/dCas9 system was activated, ear fibroblasts successfully expressed VASA gene. The results suggest that VASA gene plays a potential function in sheep testicular development and spermatogenesis, and the VASA gene knock-in vector can be constructed in vitro through the CRISPR/Cas9 system. Our results provided effective research tools for further research of germ cell development and differentiation.
Male ; Animals ; Sheep/genetics* ; CRISPR-Cas Systems/genetics* ; Gene Knock-In Techniques ; RNA, Guide, CRISPR-Cas Systems ; Plasmids ; Germ Cells

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Escherichia coli ; genetics ; Gene Knock-In Techniques ; methods ; Gene Knockout Techniques ; methods ; Humans ; Lac Operon ; genetics ; Plasmids ; genetics ; Recombination, Genetic

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Luciferases ; genetics ; Recombination, Genetic ; genetics

Alleles ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; genetics ; Embryonic Stem Cells ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Genetic Engineering ; methods ; Genomics ; Mice

As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage.
Acetylation ; Cells, Cultured ; DNA Damage ; Embryonic Stem Cells ; physiology ; Fibroblasts ; physiology ; Gene Expression Regulation ; Gene Knock-In Techniques ; Humans ; Protein Processing, Post-Translational ; Protein Stability ; Transcription, Genetic ; Tumor Suppressor Protein p53 ; physiology