Zona Pellucida Laser Puncturing for Embryo Gene Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The zona pellucida, a protective barrier around fertilized eggs, poses a challenge for viral-based gene delivery methods like lentiviruses and adeno-associated viruses, as chemical weakening or removal of the zona can impair embryonic development.
Innovation Solution
The methods involve manipulating the zona pellucida of denuded zygotes to increase permeability by either enzymatic removal or puncturing 3 to 5 holes using a laser, allowing for the efficient transfer of nucleic acids into the zygote.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical methods are used to weaken or remove the zona pellucida to facilitate viral-based gene delivery, then nucleic acid delivery efficiency is improved, but embryonic development is impaired
Solution Approach 1:
The patent replaces chemical methods with a mechanical approach by using a laser to create micro-holes in the zona pellucida. This mechanical/physical method allows viral vectors to penetrate the barrier without exposing the embryo to chemical agents that could harm development, thus resolving the contradiction between delivery efficiency and developmental safety
Solution Approach 2:
The laser-created micro-holes serve as an intermediary structure that facilitates viral vector penetration while maintaining the integrity of the zona pellucida. The holes are precisely controlled in size and number (3-5 holes) to allow sufficient viral entry while preserving the protective function of the zona, thereby enabling efficient gene delivery without compromising embryonic development
2Productivity
If the zona pellucida is completely removed to allow nucleic acid passage, then transfection efficiency is improved, but zygote vulnerability and adhesion increase
Solution Approach 1:
Instead of uniformly removing the zona pellucida, the patent applies local modification by creating specific micro-holes (3-5 holes) at controlled locations. This localized approach maintains the protective quality of the zona in most areas while providing specific pathways for nucleic acid entry, thus improving transfection efficiency without increasing overall vulnerability
Solution Approach 2:
The patent uses partial action by creating only 3-5 micro-holes rather than completely removing the zona pellucida. This partial penetration is sufficient to allow viral vector entry and achieve high transfection efficiency, while preserving enough of the zona structure to maintain zygote protection and prevent excessive adhesion
3Productivity
If single-cell embryo microinjection is used for zona penetration, then gene delivery effectiveness is improved, but operational complexity and cost increase
Solution Approach 1:
The patent replaces the complex mechanical microinjection process with a simpler laser-based approach. The laser can be precisely controlled to create micro-holes without requiring skilled technologists to manually penetrate the zona, thereby maintaining high gene delivery effectiveness while significantly reducing operational complexity and cost
Solution Approach 2:
The laser system is capable of self-positioning and self-regulation to create the appropriate micro-holes in the zona pellucida. The system automatically controls the number (3-5 holes) and size of holes based on pre-programmed parameters, eliminating the need for skilled manual operation and reducing dependency on technologist expertise
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables high transfection rates of mammalian zygotes with nucleic acids of interest, such as DNA or RNA, while maintaining zygote viability, thus overcoming the limitations of existing methods.
Implementation Method 1
puncturing 3 to 5 holes in the zona pellucida using a laser
Implementation Method 2
removal of the zona pellucida using enzymatic digestion
Data Source
AI summary
This disclosure relates to methods of transfecting or transducing mammalian zygotes with a nucleic acid of interest (such as e.g., ancient DNA). The methods generally include manipulating the zona pellucida of a denuded zygote to increase permeability of the zona pellucida to a nucleic acid of interest. In certain embodiments, the manipulating of the zona pellucida of the denuded zygote includes removing the zona pellucida with enzymatic treatment (with e.g., acid Tyrode, collagenase, hyaluronidase, or pronase). In other embodiments, the manipulating of the zona pellucida of the denuded zygote includes puncturing 3 to 5 holes in a zona pellucida of a denuded zygote using a laser; and contacting the nucleic acid of interest with the denuded zygote under conditions allowing transfection to generate a transfected denuded zygote. The nucleic acid of interest may be on a vector such as a recombinant viral vector.