Knockdown

Gene knockdown has become a commonly used approach to perform experiments in cell cultures and more recently transgenic animals. It has developed into a routine method to assess gene function in glycoengineering in a fast and easy manner. Powered by our advanced experimental technology platform and professional technical personnel, Creative Biolabs offers you a comprehensive set of gene knockdown services in cell line glycoengineering and give our global clients a guideline for glycoengineering project progression.

Background of Gene Knockdown

Different genetic techniques are in operation to investigate the functioning of genes within the living biological systems. Gene knockdown is an experimental procedure where this procedure reduces or suppresses the expression of a particular gene or genes of an organism. RNA interference (RNAi) has become a widely used approach to perform gene knockdown experiments in cell cultures and more recently transgenic animals. Gene silencing is the common definition for gene knockdown. Two types of small ribonucleic acid (sRNA) molecules, small interfering RNA (siRNA) and micro-RNA (miRNA) are central to RNAi. The primary use of this strategy is to investigate the role of the particular gene in the biological system.

Fig.1 Biological principles of RNAi. Fig.1 Principle of RNAi.^{1, 3}

Application in Glycoengineering

Many experimental cases demonstrate the effectiveness of gene knockdown or combinational genetic manipulation in glycosylation engineering. Engineered Chinese hamster ovary (CHO) cells with GnT-IV and -V overexpression and GalT4 knockdown express a highly glycosylated human chorionic gonadotropin (hCG) hormone. Besides, double siRNA knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in CHO cells provide a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC. Protein sialylation was enhanced in the whole cell process after knocking down Neu3 gene expression in CHO cells. Additionally, RNAi-mediated knockdown approach was used to reduce plant-specific glycosyltransferase activities. Similar experiments are also performed in other glycoprotein production systems. For example, the engineered yeast of a Saccharomyces cerevisiae strain capable of producing a glycoprotein with humanized Man5GlcNAc2 N-linked oligosaccharides by disrupting PMT1 and PMT2 has been generated.

Features of Our Knockdown Services

RNAi technologies
siRNA or shRNA design and synthesis service
siRNA or shRNA vector construction service
mRNA/ protein expression test
Multiple engineered CHO cell lines
Perfect after-sale service system
Therapeutic glycosylated antibodies with a higher ADCC function
Cost-effective and time-saving

Creative Biolabs utilizes many different RNAi technologies to achieve robust knockdown in cell line glycoengineering. Our gene knockdown platform allows high-level expression of shRNA and miRNA that will help our clients in various scientific research. The optimized and validated technology systems are designed to minimize and eliminate the trial-and-error phase and help you get answers faster with fewer errors. Our scientists are pleased to tailor the best-fit proposal to meet your specific demands. Please feel free to contact us for more details.

Published data

Plant cells have several advantages in the production of recombinant proteins, but there are limitations in the production of therapeutic glycoproteins according to their glycosylation mode. In this study, Agrobacterium-mediated transient expression was performed in plant cell suspension cultures. The researchers knocked down two enzymes in Nicotiana benthamiana to reduce the number of recombinant proteins containing plant-specific glycans. The results showed the production of an anthrax decoy-Fc fusion protein. N-glycosylation analysis of the protein showed a significant reduction in plant-specific N-glycans. The stability, efficacy, and safety of therapeutic glycoproteins are associated with glycosylation. This study provides us with data to support the design and optimization of plant cell glycosylation pathways and the use of plant cells for the production of therapeutic glycoproteins.

Fig.2 Characterization of anthrax decoy-Fc fusion protein N-glycosylation. Fig.2 N-glycosylation analysis of produced glycoproteins.^{2, 3}

FAQs

Q1: What are the advantages of RNAi technology?

A1: RNAi is a technology that utilizes complementary short RNAs to reduce the expression of specific genes. Generally, this effect can be induced at the transcriptional level by transcriptional silencing, or post-transcriptionally by mRNA cleavage or translational repression. With the advantages of high efficiency, simplicity, and specificity, RNAi technology is a powerful tool for studying gene functions, glycosylation pathways, and the production of recombinant glycoproteins.

Q2: What is the difference between siRNA and shRNA?

A2: siRNA has high sequence specificity and can cause rapid degradation of the corresponding mRNA after cell transfection. shRNA is transfected into cells by viruses or plasmids, and transcribed in the nucleus of cells to generate shRNA with a hairpin structure. Among them, viruses are more efficient in infecting cells. In comparison, siRNAs are easier to manipulate and work quickly, while shRNAs are more stable and long-lasting. Some factors need to be considered when choosing between siRNAs and shRNAs.

Customer Review

Good Knock-down Effect
"Creative Biolabs was very good at using various gene editing strategies to regulate glycosylation pathways. After understanding our research objectives, their researchers responded quickly and facilitated the production of recombinant glycoproteins by knocking down the expression of the relevant genes. The quality aspect of the service they provided exceeded our expectations."

Stable Knockdown
"Creative Biolabs' team of experts demonstrated exceptional knowledge and professionalism in the field of genetic glycan engineering. They analyzed the glycosylation pathway in detail after defining the experimental objectives and achieved a stable knockdown in the cell line. The whole process was efficient and smooth. If the opportunity arises, we would subsequently collaborate with them again to produce recombinant glycoproteins."

References

Isenmann, Marie, et al. "Basic principles of Rna interference: Nucleic acid types and in vitro intracellular delivery methods." Micromachines 14.7 (2023): 1321.
Sukenik, Sara C., et al. "Transient recombinant protein production in glycoengineered Nicotiana benthamiana cell suspension culture." International journal of molecular sciences 19.4 (2018): 1205.
Distributed under Open Access license CC BY 4.0, without modification.

For Research Use Only.