H19 and IG DMR Knockout in Androgenetic Stem Cells
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Solution Overview
Problem
The low birth rate and rapid decline in viability of semi-cloned mice produced using androgenetic haploid embryonic stem cells (AG-haESCs) due to abnormal imprinting status, particularly the abnormal expression of imprinted genes like H19 and Igf2, hinder efficient genome-wide loss-of-function genetic screening in mammals.
Innovation Solution
Knocking out the differentially methylated regions (DMRs) of the H19-Igf2 and Dlk1-Dio3 imprinted clusters in AG-haESCs, designated as DKO-AG-haESCs, to establish characteristics resembling round spermatids, which when combined with oocytes, result in a higher birth rate and stable production of genetically modified semi-cloned mice, enabling effective gene mutation and screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If AG-haESCs are used for producing semi-cloned mice, then genetic modification capability is improved, but birth rate and viability are severely reduced
Solution Approach 1:
The invention changes the epigenetic parameters of AG-haESCs by knocking out specific DMRs (H19 DMR and IG-DMR) to alter the imprinting status. This parameter change resolves the contradiction by making the cells more suitable for producing viable semi-cloned mice while maintaining their genetic modification capability. The DMR knockouts modify the methylation status and gene expression patterns, thereby improving reliability without sacrificing adaptability.
Solution Approach 2:
The invention extracts and removes the problematic differentially methylated regions (DMRs) from the AG-haESCs. By taking out the H19 DMR and IG-DMR elements that cause abnormal imprinting, the invention eliminates the harmful effects on birth rate and viability while preserving the essential genetic modification functions of the cells.
2Quantity of substance
If long-term culture of AG-haESCs is performed, then cell expansion is achieved, but birth rate of semi-cloned mice declines rapidly
Solution Approach 1:
The invention performs preliminary action by knocking out the DMRs in advance during cell culture, before the cells are used for producing semi-cloned mice. This preliminary modification of the epigenetic status ensures that even after long-term culture and expansion, the cells maintain their ability to produce viable offspring, preventing the rapid decline in birth rate that normally occurs with prolonged culture.
3Stability of the object's composition
If imprinted genes are expressed abnormally, then development of uniparental embryos is hindered, but this is the natural state of AG-haESCs
Solution Approach 1:
The invention changes the imprinting parameter status by selectively knocking out specific DMRs (H19 DMR and IG-DMR) rather than attempting to restore all imprinted genes. This selective parameter change resolves the contradiction by creating a modified imprinting status that is stable in the cells but permissive for embryo development, thereby improving ease of operation without completely altering the uniparental nature of AG-haESCs.
Data Source
Figure 1A~1F
Figure 1G~S1C
Figure S1D~S1F
AI summary
The present invention relates to an AG-haESCs in which H19 DMR and IG-DMR are knocked out, a method for preparing the AG-haESCs, and use of the AG-haESCs in constructing a genetically modified semi-cloned animal and a library of a genetically modified semi-cloned animal. The AG-haESCs is capable of obtaining characteristics resembling a round spermatid, and upon injection into an oocyte, a viable SC mouse is stably obtained. The present invention is capable of being effectively used in multi-gene genetic manipulation, advancing the acquisition of animals with multiple genetic modifications.