Rat Genome Modification via Large Targeting Vectors
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The use of rats as animal models for human diseases is limited due to the unavailability of germline-transmittable pluripotent rat cells that can sustain pluripotency after genetic modifications and the lack of efficient targeting technologies for introducing or deleting large genomic DNA sequences in rat cells.
Innovation Solution
Methods are provided for modifying rat genomic loci using large targeting vectors (LTVEC) and bacterial homologous recombination (BHR) to introduce targeted genetic modifications that can be transmitted through the germline, involving the use of pluripotent rat cells and prokaryotic cells to achieve precise changes in the rat genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional targeting technologies are used in rat cells, then the process is simpler, but the ability to introduce or delete large genomic DNA sequences is insufficient
Solution Approach 1:
The patent segments the targeting process into two distinct phases: (1) bacterial homologous recombination in prokaryotic cells to assemble large targeting vectors with precise genomic modifications, and (2) introduction of the constructed vector into pluripotent rat cells. This segmentation allows complex large-scale genomic modifications to be achieved by breaking down the process into manageable steps that can be performed in optimized systems.
Solution Approach 2:
The patent uses prokaryotic cells as an intermediary system to perform homologous recombination and construct large targeting vectors before introducing them into rat cells. This intermediary approach leverages the efficient recombination capabilities of bacterial systems to prepare complex genetic modifications, which would be difficult to achieve directly in mammalian cells.
2Adaptability or versatility
If serial electroporations are performed for genetic modifications, then multiple genes can be modified, but pluripotency is lost
Solution Approach 1:
The patent performs preliminary construction of large targeting vectors in prokaryotic cells using homologous recombination before introducing them into pluripotent rat cells. This preliminary action allows multiple genetic modifications to be pre-assembled in a single vector, enabling all desired modifications to be introduced in one electroporation event rather than through multiple serial electroporations that would compromise pluripotency.
3Reliability
If traditional gene targeting methods are used, then the methodology is well-established, but the efficiency of generating germline-transmittable modifications is low
Solution Approach 1:
The patent replaces traditional mechanical/electrical methods (serial electroporations) with a molecular biology-based approach using bacterial homologous recombination to construct targeting vectors. This substitution leverages the natural homologous recombination machinery of bacteria to achieve precise large-scale genomic modifications, which are then introduced into rat cells with higher efficiency and reliability for germline transmission.
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
These methods enable precise targeted genetic modifications in rat cells, allowing for the expansion of disease modeling and therapeutic agent validation, facilitating quicker and more efficient discovery and validation processes.
Implementation Method 1
Compositions and methods for modifying a rat genomic locus of interest via bacterial homologous recombination (BHR) in a prokaryotic cell
Data Source
AI summary
Compositions and methods are provided for modifying a rat genomic locus of interest using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Compositions and methods for generating a genetically modified rat comprising one or more targeted genetic modifications in their germline are also provided. Compositions and methods are provided which comprise a genetically modified rat or rat cell comprising a targeted genetic modification in the rat interleukin-2 receptor gamma locus, the rat ApoE locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The various methods and compositions provided herein allows for these modified loci to be transmitted through the germline.