2-Cell Tetraploid Embryo Aggregation for Mouse Model Production
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Solution Overview
Problem
Current methods for preparing animals, particularly mice, using the tetraploid complementation technique face challenges such as low fetal birth rates and inefficient gene editing, limiting their practical application in drug screening and research.
Innovation Solution
A method involving the aggregation of a 2-cell tetraploid embryo with embryonic stem cells to form a chimeric embryo, which is then implanted into a pseudopregnant animal, using a specific composition and fusion solution to enhance fusion efficiency and stability, thereby improving birth rates and facilitating gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional tetraploid complementation technique is used to prepare animals, then animal models can be established, but the fetal birth rate is low and gene editing efficiency is poor
Solution Approach 1:
The invention segments the traditional single-step tetraploid complementation process into multiple optimized stages: (1) preparation of diploid embryos with enhanced developmental potential, (2) fusion with embryonic stem cells to create tetraploid complementation embryos, (3) selective culture and screening of high-quality embryos. This segmentation allows each stage to be optimized independently, resulting in significantly improved fetal birth rates while maintaining animal model establishment capability
Solution Approach 2:
The invention applies preliminary action by pre-selecting and pre-culturing diploid embryos to ensure they have optimal developmental potential before fusion. Embryos are screened for morphological quality and developmental stage appropriateness beforehand, and embryonic stem cells are prepared and validated prior to fusion. This preliminary preparation ensures that only high-quality embryos proceed to the fusion step, thereby improving overall efficiency and fetal birth rates
2Reliability
If traditional tetraploid complementation technique is used, then animal models can be prepared, but the process requires extensive time and resources
Solution Approach 1:
The invention changes key parameters of the traditional process: (1) optimizes the developmental stage selection for diploid embryos (using 2-cell to 8-cell stage embryos), (2) adjusts fusion conditions and embryonic stem cell ratios, (3) modifies culture medium compositions and incubation conditions. These parameter optimizations accelerate embryonic development and improve survival rates, reducing the overall time required from embryo preparation to viable animal model generation
Solution Approach 2:
The invention employs copying by creating standardized protocols and using cryopreserved embryonic stem cell lines that can be repeatedly used without loss of effectiveness. High-quality diploid embryo preparation methods are standardized and can be replicated across multiple experiments. This copying approach eliminates the need to re-optimize conditions for each experiment, significantly reducing time and resource requirements while maintaining consistent animal model preparation quality
3Ease of manufacture
If conventional animal preparation methods are used, then basic animal models can be obtained, but gene editing capability is limited
Solution Approach 1:
The invention merges two previously separate processes: (1) tetraploid complementation for animal model generation, and (2) gene editing using embryonic stem cells. By combining CRISPR/Cas9 or other gene editing systems directly into the embryonic stem cells used for complementation, the invention enables simultaneous achievement of both animal model production and precise gene editing in a single integrated workflow, greatly enhancing versatility without compromising ease of manufacture
Solution Approach 2:
The invention creates a composite biological system combining diploid embryos, embryonic stem cells with integrated gene editing machinery, and tetraploid complementation mechanisms. This composite approach allows the system to perform multiple functions: the diploid embryos provide developmental potential, the embryonic stem cells contribute gene editing capability through pre-integrated transgenes or CRISPR components, and the tetraploid complementation ensures proper placental development. The result is a versatile platform that produces gene-edited animal models efficiently
Data Source
AI summary
Provided are an animal preparation method and use thereof. The method includes aggregating a tetraploid embryo with embryonic stem cells to form a new reconstructed embryo or chimera embryo, the tetraploid embryo being a tetraploid embryo developed to 2-cell stage. By aggregating a 2-cell tetraploid embryo with embryonic stem cells, problems of poor efficiency and poor stability of mice preparation using the tetraploid complementation technique as well as low efficiency when embryonic stem cells from pure line mice are used are alleviated, the birth rate of mice is improved to a level close to that of normal embryo transplantation, and embryos and adult mice can be directly prepared from stem cells for phenotypic research.