Chimeric Embryo Production for Knockout Large Mammal Gametes
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Solution Overview
Problem
The challenge lies in the difficulty of mass-producing knockout individuals in large animals due to low fertility, spontaneous abortion, and high mortality rates associated with somatic cell nuclear transfer techniques.
Innovation Solution
The method involves creating a chimeric embryo by transplanting pluripotent cells from one non-human large mammal or fish into an embryo with a knocked-out nanos3 gene, allowing germ cells to be complemented and producing gametes with specific genetic characteristics, enabling steady production of knockout animals through artificial insemination or in vitro fertilization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If somatic cell nuclear transfer technique is used to produce knockout large animals, then gene knockout can be achieved, but low fertility, spontaneous abortion, stillbirth, and high mortality occur frequently
Solution Approach 1:
The invention divides the embryo into two functional components: the body cells derived from knockout embryos (providing the desired gene knockout trait) and the germ cells derived from normal embryos (providing reproductive capability). This segmentation allows the knockout trait to be present in somatic cells while normal germ cells ensure successful reproduction, resolving the contradiction between gene knockout accuracy and reproduction success rate.
Solution Approach 2:
The invention uses normal embryos as an intermediary to provide healthy germ cells that can compensate for the reproductive defects in knockout embryos. The normal embryos serve as a bridge, donating germ cells through chimeric embryo formation, thereby enabling knockout animals to reproduce successfully without compromising the gene knockout trait.
2Quantity of substance
If conventional cloning methods are used for mass production, then genetic characteristics can be replicated, but production efficiency is low due to high mortality and abortion rates
Solution Approach 1:
The invention performs preliminary action by creating chimeric embryos that already contain both knockout somatic cells and normal germ cells before implantation. This preliminary configuration ensures that the resulting animals will have both the desired knockout trait and normal reproductive capability from birth, eliminating the need for subsequent corrective procedures and enabling efficient mass production of knockout individuals.
Solution Approach 2:
The invention changes the fundamental parameter of embryo composition from homogeneous (all cells from one source) to heterogeneous (chimeric composition with cells from two sources). By altering the cellular origin parameters, the invention simultaneously achieves gene knockout in somatic cells and normal germ cell function, thereby increasing both the quantity of knockout individuals and overall productivity.
3Ease of manufacture
If ES cells are used for gene knockout in mice, then knockout can be achieved easily, but this method is not applicable to large animals with unestablished ES cells
Solution Approach 1:
The invention copies the successful mouse knockout strategy (using embryonic cells for gene modification) and adapts it to large animals by using embryonic cells at the cleavage stage instead of established ES cells. This copying approach allows the same fundamental principle to be applied across species boundaries, achieving both ease of manufacture and broad species applicability.
Solution Approach 2:
The invention creates a universal method that works across different species by using a common approach: injecting pluripotent cells into cleavage-stage embryos. This multi-functional technique can be applied to mice, cattle, pigs, and other large animals regardless of whether species-specific ES cell lines are established, thereby achieving both ease of manufacture and wide species applicability.
Data Source
AI summary
Disclosed is a novel means which makes it possible to steadily mass-produce knockout individuals even in large animals. The method of the present invention is a method for producing a non-human large mammal or fish (non-human animal) that produces gametes originating in a different individual, and comprises transplanting at least one pluripotent cell derived from a second non-human animal into an embryo derived from a first non-human animal, said embryo being at a cleavage stage and having a genome in which a function of nanos3 gene is inhibited, to prepare a chimeric embryo, and allowing said chimeric embryo to develop into an individual.