Biocytogen

RenMab Mouse Antibodies

Biocytogen’s RenMab™ Mouse is an antibody-humanized mouse with the most complete antibody genes replacement in the world, which has been designed to greatly simplify and de-risk the antibody development process. In the RenMab™ Mouse,

1. The parts of the genes that encode for antibody variable domains of H and κ chains are replaced in situ by human sequences using a unique chromosome engineering technique, while the constant domains and critical regulatory elements remain murine. 

2. The RenMab™ Mouse has a normal immune system comparable to that of the wild-type mouse. Because of the higher diversity of its antibody genes, the RenMab™ Mouse has a higher potential for antibody drug development.

3. The RenMab™ Mouse is the ideal platform for researchers to generate fully human antibody-based drugs with strong specificity, high affinity, and diversity.

Biocytgoen offers an array of double and triple humanized mice for combination therapy in vivo efficacy studies.

See the link below for further information: https://biocytogen.com/products/humanized-immune-checkpoint-mice/

The Immune-deficient B-NDG mouse model (NOD-Prkdcscid IL2rgtm1/Bcgen) was independently designed and generated by Biocytogen. B-NDG mice are generated by deleting the IL2rg gene from NOD-scid mice with severe immunodeficiency phenotype. Lacking mature T cells, B cells or functional NK cells, and displaying cytokine signaling deficiencies, this mouse model has the highest degree of immunodeficiency and thus is most suitable for engraft and growth of human hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs) and human tumor cells or tissues.

• NOD-scid (non-obese diabetes, severe combined immunodeficiency) genetic background: mice of NOD genetic background and with Prkdc (protein kinase DNA-activated catalytic) knockout. Functional T cells, B cells and the complement system in these mice are lost, and the activity of NK cells is greatly weakened.
• IL2rg null: the gamma chain of Interleukin-2 receptor (IL-2R γc, also called CD132) is on the mouse X chromosome, and is the common receptor subunit of cytokines IL2, IL-4, IL-7, IL-9, IL-15 and IL-21 with important immune functions. After IL2r is knocked out, mouse immunity function is greatly weakened, activities of NK cells, which are almost completely lost.
• Prkdc null (DNAPK, scid): Prkdc (protein kinase DNA-activated catalytic) null mutation is characterized by significantly deficient of functional T cells and B cells, and an absence of lymphocytes, recapitulating severe combined immunodeficiency (scid) in human patients.

Advantages of B-NDG Mice

• Model currently holds the highest degree of immunodeficiency among all immunodeficiency models
• Longer lifespan than NOD-scid mice; 1.5 years on average
• Minimal to absent rejection of human-derived cells and tissues
• More efficient for CDX and PDX model generation
• No B lymphocyte leakage

Major Applications

• Human-derived cell or tissue engraftment
• Tumor and tumor stem cell research
• ES and iPS cell research
• Hematopoiesis and immunology studies
• Human infectious disease studies
• Development of humanized models

Biocytogen offers industy leading models of humanized immue-checkpoints and cytokines/cytokine receptors. Please see our website for more information:

https://biocytogen.com/products/humanized-immune-checkpoint-mice/

https://biocytogen.com/products/humanized-cytokines_mice/

Definition: A mouse model with a functional human gene, cell, tissue, or organ, which is used as living body substitution model for human disease study. This service refers to the model with the functional human gene.

Advantages: the human gene is expressed in the mouse body using the promoter and regulation region of the mouse gene.

Disadvantages: to ensure that the human gene is physiologically expressed and regulated normally in the mouse genome, the sequence and structure information of the genes and proteins should be analyzed in depth before designing, and comprehensive risk assessments should be built.

Principle

A humanized mouse model with a functional human gene is constructed by replacing part or all of the mouse gene with the human gene. The expression of the human protein in the mouse body is the same as the mouse protein’s expression, but the protein or functional region(s) of the mouse protein is not expressed in any cells or tissues.

Application

• Human gene expression and functional studies
• Human disease studies, such as AIDS, cancer, infectious diseases, inflammatory diseases, human degenerative diseases, and blood diseases, etc.
• Preclinical drug study
• Compound safety evaluation

Brief introduction of the technology

TALEN (Transcription Activator-Like Effector Nuclease) is another high-efficiency genome modification tool. Its high efficiency, strong specificity, low missing rate, and simple design makes the technology widely used for gene knockout and genome modification at the cell and in vivo levels.

Service process

BIOCYTOGEN provides services for mouse model construction in one step, including model designing, construction, and genotypic identification service

• Project feasibility evaluation and model design

BIOCYTOGEN possesses a senior technical team specializing in comprehensive analysis of project feasibility, and provides a free and professional model design service. The services include:

Ÿ Define the purpose for constructing the model
Ÿ Analyze the target gene’s structure and sequence information
Ÿ Analyze existing data
Ÿ Design all possible model construction schemes
Ÿ Analyze the advantages and risks of all model construction schemes
Ÿ Accurate prediction of project implementation time and quotation

• Design and construction of TALEN

• Pronuclear injection

Ÿ Pronuclear injection of the mRNA (TALEN) into the mouse zygotes

• Obtain the mouse of generation F0 and breeding

Ÿ Recipient female mouse feeding and the birth of chimeric mouse
Ÿ Mating of chimeric mouse and wild mouse

• Obtain the phyla-genetic hybrid mouse of generation F1

Ÿ Birth of generation F1 mice
Ÿ Ensure the phyla heredity by identification of generation F1 mouse tail genotype

Definition:

Use the homologous recombination technology to replace all the exons, several important exons, or function domains of a target gene using a section of exogenous DNA sequence, and obtain a mouse model where all of the tissues and cells during all developmental stages (from 1-cell embryo to adult stage) do not express the gene.

Advantages and disadvantages:

• Advantages: the design of the targeting strategy and construction of the target vector is simple
• Disadvantages: the knockout of the target gene may cause embryonic death or expression dysregulation of other genes. The conditional knockout design strategy can be used if this risk is predicted to exist.

Application:

• Physiological or pathological function study of a gene on the whole-body level
• Drug target affirmation
• Drug toxicological study

Principle:

The conventional knockout realizes the target gene knockout by replacing one or more exons of the target gene using the screening gene neomycin (Neo). The knockout occurs in all of the tissues and cells in the body, which includes the risk of embryonic death. This risk can be avoided by a conditional knockout strategy.

*Pharmacological and pharmacodynamic test for CDX (Cell line-Derived Xenograft) tumor model* CDX (Cell line-Derived Xenograft) is a well established model for the research and development of tumor drugs. The model is easy to establish and has excellent repeatability, so it is still under high demand for the research and development of tumor drugs.

Cell line models are established by inoculating passage tumor cells in vitro, and culturing them into the body of immune deficient mice

Available services:

• Different cell lines for tumor models according to the customer’s requirements for research and development;
• establishment of subcutaneous tumor model and orthotopic tumor model or metastatic tumor model;
• in vivo pharmacological and pharmacodynamic service for the corresponding models.

Targeting Vector Construction Services

1. ES Cell based targeting vector: (1-2 months) Including analysis of gene structure, screening strategy design, targeting vector construction and confirmation (DNA sequencing verification). Southern blot strategy and probe design will also be provided.

• CRISPR/Cas9 based sgRNA/Cas9 vector (1-2 months): Including analysis of gene structure, screening strategy design; construction and confirmation of CRISPR/Cas9 plasmids, sgRNA activity testing (selected the best activity one among 4-6 sgRNAs)
• CRISPR/Cas9 based donor plasmid (DNA template for cKO/KI): (1-2 months) Including analysis of gene structure, screening strategy design; targeting vector construction and confirmation (DNA sequencing verification)
• Sub-cloning plasmid: (2-3 weeks)

• Rosa26 locus gene knockin mouse model
• Reporter/tag mouse model
• Conditional point mutation mouse model-tissue specific
• Conditional point mutation mouse model-induced
• Conventional point mutation mouse model
• Humanized mouse model
• Knockin mouse model
• Knockout first, conditional ready mouse model
• Conventional knockout mouse model
• Gene Targeting Technology Based on Embryonic Stem Cell (ESC/HR)

Definition: mouse models with single or multiple base changes or an exogenous gene introduced into the target gene position.

Principle: Knockins introduce specific mutation(s) or an exogenous gene in the target gene position. For example, this method can introduce point mutation(s), which can simulate a human genetic disease, in the target gene. It can also be used to introduce a reporter gene (such as EGFP, mRFP, mCherry, mYFP or LacZ, etc.) or functional cDNA (such as Cre and Dre, etc.) to be expressed in a specific locus by homologous recombination, thereby making the expression of the reporter gene or other cDNA accordant with the expression of the target gene. The knockout and knockin occurs at the same time when the reporter gene or cDNA replaces the mouse gene.

Using the EGE system, EGFPand mCherrygenes are knocked into the ACTB and LMNB1 translation initiation sites, respectively, in the rodent C6 cell lines.

By using flow cytometry analysis, we have found that the CRISPR/Cas9 mediated gene EGFP-ACTB knockin in efficiency is only 1.91% in U2OS cells while the efficiency is 15.02% by using EGE system, which shows an 8-fold increase. In C6 cell line, the EGFP-LMNB1 knockin efficiency increased from 0.19% to 3.6% by using EGE system, which is about a 19-fold increase.