Mouse Cell Line Authentication via Multiplex STR Profiling
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for authenticating mouse cell lines lack the resolution to differentiate between individual mice of the same subspecies, and existing techniques using microsatellite or SNP markers are not sufficient for unique identification at the individual level, especially due to high mutation frequencies and conserved markers between inbred mice strains.
Innovation Solution
A PCR assay utilizing tetranucleotide repeats and specific STR marker loci, including 18-3, 4-2, 6-7, 9-2, 15-3, 6-4, 12-1, 5-5, and X-1, to generate unique profiles for individual mouse cell lines, with primer pairs designed to amplify these loci in a multiplex reaction, providing stable results even at high passage numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If microsatellite markers (dinucleotide repeats) are used for mouse cell line authentication, then amplification can be achieved, but noisy stutter and high mutation frequencies result in reduced measurement precision
Solution Approach 1:
The patent changes the repeat unit parameter from dinucleotide (CA) to tetranucleotide (GATA) repeats. This parameter change reduces stutter noise and mutation frequency, thereby improving both measurement precision and reliability of cell line authentication
2Measurement precision
If SNP markers are used for mouse cell line identification, then conserved markers can be detected, but resolution to differentiate between individual mice of the same subspecies is insufficient
Solution Approach 1:
The patent segments the genetic analysis into multiple independent STR loci (18-3, 4-2, 6-7, 9-2, 15-3, 6-4, 12-1, 5-5, X-1) distributed across different chromosomes. Each locus provides independent discriminatory power, and the combination of multiple loci achieves high differentiation resolution that SNP arrays cannot provide
3Measurement precision
If species-specific primers are used to determine cell line origin, then species identification can be achieved, but specificity to identify at the individual level is insufficient
Solution Approach 1:
The patent transitions from species-level identification (one dimension) to individual-level identification by adding chromosomal location and allele pattern dimensions. The STR profiles include chromosomal location data and specific allele combinations that are unique to each individual mouse, enabling identification at the individual level while maintaining species specificity
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
The assay provides stable and unique genetic profiles for mouse cell lines, capable of distinguishing between individual mice and detecting human cell line contaminants, offering a reliable method for authentication with high sensitivity and specificity.
Implementation Method 1
A PCR assay utilizing tetranucleotide repeats and specific STR marker loci, including 18-3, 4-2, 6-7, 9-2, 15-3, 6-4, 12-1, 5-5, and X-1, to generate unique profiles for individual mouse cell lines
Data Source
AI summary
A multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.