ES Cell Screening (PCR)

Screening homologous recombination in ES cells

To screen and confirm homologous recombination (HR) in Embryonic Stem (ES) cells, extracted DNA from individual ES cells is first genotyped using a Loss-of-Allele (LOA) assay. LOA is a quantitative TaqMan® qPCR detecting the loss of one region of the native target due to correct homologous recombination with the gene targeting vector. In HR positive clones, we expect to detect one copy of this region in the recombined allele and two copies in the non-recombined, or wild type, allele.  Assays are developed and validated before samples are processed, in triplicate, on an automated AB 7900HT in 384 well plates. Samples are then analyzed by relative cycle threshold method. Once the initial candidates are identified and further expanded from 96 well plates to 6 well plates in the ES cell laboratory, the selected clones are then confirmed with LOA, selection cassette copy number analysis (ensuring there is only one copy of the selection cassette and not a targeted as well as random integrant), LoxP check for conditional alleles (ensuring both LoxP sites remained intact during homologous recombination) and Long Range PCR across both 5’ and 3’ arms of homology (PCR from vector/non genomic DNA to outside of the long arm of homology).  In addition, we also offer high throughput screening to detect successful FLP/Cre recombinase mediated excision of the selection cassette(s).

Mouse Colony Genotyping

Rapid and accurate mouse genotyping

The MBP offers a variety of services for mouse genotyping, customized to meet the needs of our clients. We apply a high throughput platform utilizing integrated robotics and LIMS and perform standard simplex or multiplex PCR in addition to quantitative TaqMan® qPCR. Common universal target TaqMan® probes include Neo, LacZ, Puro, eGFP, Tomato, Cre and Dre recombinase, and Luciferase. In addition, we routinely design, customize, and validate TaqMan® and standard PCR assays for unique mutant mouse lines. Once an assay is designed and validated, and samples and request form received, genotyping results are typically reported both online and emailed to the client in less than 5 business days and with 99.7% accuracy.

Taqman qPCR Gene Expression

Quantification of Transgenic, KI, KO and ENU mutants or Wild type mouse gene expression

Gene Expression for most tissues types is available using qPCR quantification for Transgenic, Knock-in (KI), Knockout (KO), and ENU mutants.  We specialize in using the relative Ct method to compare the expressed gene of interest to an internal reference gene such as GAPDH or Beta-actin and further quantify the reference sample using the ΔCt of the tissue assayed compared to a reference ΔCt of a control which is reported as a ΔΔCt.  Briefly, tissues (normally stored in RNAlater) are homogenized and tRNA extracted robotically using Qiagen RNEasy plate columns. RNA concentration from each tissue is confirmed by nanodrop and RNA from each tissue is converted into a cDNA library via high capacity cDNA amplification using random hexamers for long term storage and qPCR quantification.  Controls samples include a reference tissue and a negative RT enzyme control.  The cDNA for each tissue is assayed in quadruplicate with a reference (GAPDH or Beta-actin) as well as the target assay.  The Average ΔCt between the reference and the targets is used for ΔΔCt quantification analysis and reported in fold changes between the diagnostic samples and a reference sample.  We have a large library of standard cassettes that can be used without a Taqman® assay development fee such as LacZ, Cre, GFP, YFP, Neo.  Generally, assay design in natural genes is designed to span exon-exon junctions when applicable.  This service is extremely usefully for verification of Trangenic(TG) or Knock-in(KI) expression, in additon to verification of expression reduction knock out regions or downstream of gene-trapped alleles.    

Rapid Amplification of cDNA Ends (RACE)

Gene trapped clone confirmation

RACE is used to verify clone identity for gene trapped ES cells which result in transcriptional fusion. In these gene trapped clones, the upstream exon of the trapping cassette is identified via sequencing of the cDNA. 

Vector Integration Site Analysis (VISA)

Gene trap insertion site location

The VISA is a customized strategy for discovering the exact insertion location of a genetrap vector (usually in intron).  In general, VISA is more informative than the sequence obtained by the RACE procedure because an allele specific genotyping protocol may be developed. In addition, the VISA procedure is a genomic verification of the insertion event vs. transcriptional evidence.The VISA service includes copy# assessment to ensure a single copy/genome, selection of method for amplification and sequencing of unknown flanking region to the vector, and a validated multiplex PCR protocol that we provide in addition to the raw sequence showing the insertion junction.  In addition, we will add RACE verification of the clone (if not previously confirmed when you purchased the cell line). The VISA cost and time line is dependent on intron size of the gene trapped allele.

Southern Blot Genotyping Confirmation

Quantification of Transgenic, KI, KO and ENU mutants or Wild type mouse gene expression

Southern blot analysis reveals information about DNA identity, size, and abundance and is especially useful for targeting confirmation in ES cells when long-range PCR is problematic. This classic genotyping technique involves separating DNA fragments based on size via electrophoresis, transferring DNA to a membrane, hybridization with a labeled sequence-specific probe, washing, and finally detection of labeled DNA band(s). The MBP offers ES cell targeting confirmation via southern as an optional service.

Vector Integrity and Genomic Integrity (VIGI)

DNA integrity assessment of ES cell clones from high throughput targeting efforts (KOMP/EUCOMM)

VIGI is used to assess DNA quality, the ability to amplify large regions of DNA from a particular sample, and to confirm the 5' region of a CSD genetrap cassette. Originally created to test ES cell clone integrity for the high throughput gene targeting effort when long-range PCR reactions fail, the VIGI assay is a multiplexed long range PCR assay that validates both the DNA integrity and the ability to amplify a long range PCR band (~8kb WT) as well as verify the presence of the genetrap cassette in KOMP derived alleles from high throughput targeting efforts. 

Speed Congenics

Save up to a year and a half of breeding by screening the background genetics of your research animals and selecting those with the highest percentage of the desired background.

Speed Congenics analysis provides a percentage of the preferred background, and approximate generation number, and a recommendation of those animals to be used for the next breeding cycle. Using  marker-assisted congenic breeding strategy to produce congenic mice with >99% recipient strain genetic purity in half the time (5 generations; 1.25-1.5 years) instead of the standard 10 generations, which can take 3 years or longer.

Standard PCR Protocol Development

No genotyping protocol? We can design one for you!

Standard PCR Protocol Development service includes the genotyping design, ordering and optimization of primers needed to produce a genotyping protocol that your lab may use to genotype mice


Next-Generation Sequencing (NGS)

Next-generation sequencing enables researchers to study biological systems at a level never before possible

The Mouse Biology Program has partnered with BGI@UC Davis to offer full service genomics, transcriptomics, and epigenomics for mouse model systems. Mouse Biology Program bioinformatics systems are integrated with sample collection, nucleic acid isolation, quality assessment, library construction, and sequencing. System integration facilitates high quality downstream analysis, including sequence read quality trimming, alignment, assembly, transcript and gene counts, variant calling, ontology enrichment, and supporting statistics. The Mouse Biology Program and BGI@UC Davis aim to provide the very best sequencing services for your mouse model research.






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