ES shooting technique service

1ES shooting technique serviceTechnical Definition and Core Principles

ES shooting techniqueIt is a gene editing method based on homologous recombination of embryonic stem cells (ES), which integrates exogenous DNA at specific locations in the genome to achieve precise modifications such as gene knockout (KO), conditional knockout (CKO), gene knock in (KI), and point mutation (PM). The core principles include:

1. Homologous recombination mechanism：

• Design includesHomologous armA vector homologous to the target gene is used to insert exogenous sequences (such as screening marker genes) into the target site through homologous recombination.

2. Positive and negative screening system：

• Positive filtering tags(such as Neo ᵣ): Insert homologous regions and screen for successfully recombinant ES cells.

• Negative filter mark(such as HSV tk/DTA): located on the outer side of the homologous arm, eliminating randomly inserted cells.

3. Pluripotency of embryonic stem cells：

• The modified ES cells were injected into blastocysts and developed into chimeric mice (F0), which were then transmitted through the reproductive system to obtain a stable genetic model.

Technical positioningES targeting is the gold standard in the pre CRISPR era, especially suitable forLarge segment editing(>10 kb) andComplex gene modification(such as conditional knockout).

IIES shooting technique serviceStandardized operating procedures and technological innovation

（1） Traditional ES shooting process (7-12 months)

Key step analysis：

1. vector construction：

• The homologous arm length is usually 3-5 kb, and large fragment insertion requires a BAC vector.

2. ES cell line selection：

• Commonly used strains: 129S6/SvEvTac (high recombination efficiency), C57BL/6N (background purity).

3. Preparation of chimeras：

• The chimerism rate after blastocyst injection is about 20-70%, and it needs to be visually screened through hair color markers (such as albino hosts).

（2） Technological innovation: EPS cell targeting

EPS cellsExtended Pluripotent Stem Cells was reported by Chinese scientists in 2017, and its advantages include:

1. Efficiency improvement：

• Target efficiency reaches that of traditional ES cells10-100 times.

2. cycle reduction：

• Chimera can be obtained in one step, saving 3 months of lineage transmission.

3. Enhanced omnipotence：

• Single cell injection into blastocysts can generate 99.99% chimeras.