ES cell Gene Targeting

1. Targeted vector construction (4-6 months)

The MBP utilizes a variety of different techniques including BAC recombineering, Yeast Recombineering, Gibson Assembly Cloning, and traditional ligation to create the desired Constitutive Knockout (KO), Conditional Knockout (cKO), Knockin (KI) and Transgenic (Tg) vectors in the most efficient manner possible.

A client need only provide the gene name and the desired product, we’ll do the rest! Please visit of Molecular Biology Page for additional information on the many services available through MBP.

2. ES cell Electroporation (2 months)

Our standard electroporation service includes electroporation (with or without engineered nucleases) of a purified linearized targeting vector into JM8.N4 (C57BL.6N) or R1 (129X1 x 129S1), selection and picking of up to 96 – 284 clones and expansion of up to 12 potential positives

3. ES cell expansion (optional for outside clones 1 months)

ES cell expansion service is needed when only of a single vial of cells ( e.g., EUCOMM or outside targeting lab) is available and back-up vials are needed. In general 6-8 vials are frozen back/per clone. When a targeting vector is electroporated here at the MBP, expansion fees are included in the electroporation rate.

4. ES cell screening (PCR) (2 months including ES cell expansion time)

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).

5. Chromosome counting (1 month)

Chromosome counting is performed at the Mouse Biology Program to determine the percentage of euploid metaphase chromosomes in the targeted ES cell clones. A normal diploid mouse chromosome count is 40. We count at least 20 spreads. If 50% or more of the spreads counts total 40, the clone is considered to more likely contribute to both somatic cell chimerism and to germline transmission.

6. Flp or Cre electroporation (e.g., excise positive selection marker 2 months)

The invitro recombinase electroporation service includes vector and ES cell preparation, electroporation and, picking of up to 96 clones.

7. Microinjection (2 months)

Microinjection provides injection of at least 30-40 embryos per clone, and housing until pups are 7 weeks of age. There are no guarantees for derivation of high percent chimeric mice with basic microinjection services.

8. Germline Transmission Testing (4-6 months)

Chimera are produced when clonal mutant ES cells are injected into donor blastocysts (or morula) and resulting pups contain a mixture of both mutant and wild type (host embryo) cells. At the MBP, chimera are assessed for coat color at 10 days of age. The % coat color that is mutant cell derived is thought to correlate with the probability that mutant cells contributed to the germline (germ) cells of the chimera. Therefore, all appropriately sexed chimera determined to contain 50% or more mutant cells will be set with females. Breeding will begin when males are 7 to 9 weeks of age. When possible, each male chimera will be set with up to two females of the appropriate genetic background for GLT breeding. Most mouse Embryonic Stem (ES) cells lines are derived from male mice, this is the case for KOMP and Eucomm generated ES cells and the ES cells used by the MBP for targeting projects. Therefore, in most cases, only the male chimera are bred for Germline Transmission testing (GLT). Chimera are allowed to breed until either they produce the desired heterozygous germline pups, they are deemed nonproductive, or they produce 25 wild type pups, whichever comes first. If a male chimera is non-productive for 30-45 days, the male will be submitted for sperm collection, sperm morphological analysis and determination of suitability for IVF services. Please note that some chimera are hermaphroditic and may not produce sperm at all.

Targeted Allele Types

1. Constitutive Knockout (KO)

Permanently inactivate, or “knock out”, a gene by replacing or disrupting it in every cell of the mouse at all stages of development.

Why choose a constitutive KO?

A constitutive knockout model is a fast and cost-effective solution for preliminary study of in vivo gene function.

General Considerations:

The constitutive knockout of a target gene may be embryonic lethal and therefore will result in less flexibility for in vivo studies in adult mice. About 30% of gene knockouts can be developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. This problem is often overcome through the use of conditional mutations (e.g. a tissue specific KO or an inducible deletion of the target gene) which targets the gene knockout to a cell type that does not induce developmental lethality.

2. Conditional Knockout (cKO)

Inactivate gene expression in a specific target cell type and/or at a defined developmental stage.

Why choose a conditional KO?

Greater flexibility! The conditional approach circumvents the limitations that are usually observed with constitutive knock-out models (e.g. embryo lethality). In general, we use a Cre/loxP based strategy in which the critical exon(s) is flanked by loxP sites. Therefore, the gene will have normal, endogenous expression until Cre-mediated recombination which deletes the critical exon(s). Cre recombination will ultimately create a frameshift mutation, resulting in a premature stop and renders the gene inactive.

General Considerations:

The design process of creating a conditional knockout model requires a thorough informatics review in order to avoid disrupting endogenous gene expression of both the target and surrounding genes. In addition, the timeline for this type of project is generally longer and project costs typically higher than a constitutive knockout model since the deletion of the gene requires an additional breeding step with a tissue-specific Cre mouse line.

3. Knock-in (KI)

Insertion of a point mutation, reporter/tag, or protein coding cDNA sequence at a defined targeted location…

Why choose a Knockin Model?

Knockin mouse model technology may be used to further study gene function for a variety of methodologies ranging from a simple reporter Knockin to study the expression of a targeted gene at the transcriptional or translational level to a more precise point mutation (nucleic acid substitution or deletion) in order to further elucidate the underlying role of small nucleotide mutations in human diseases.

General Considerations:

The design process of creating a Knockin model requires a thorough informatics evaluation in order to avoid disrupting endogenous gene expression of both the target and surrounding genes. In addition, a constitutive point mutation may be lethal. In that case, the use of an inducible mutation may be required. For a reporter Knockin method, the major disadvantage is that it usually requires the endogenous gene to be replaced (knocked out) by the reporter.

4. Targeted Transgenic (TT)

Introduction of a targeted insertion of a transgene into the Rosa26 or HPRT locus…

Why choose a Targeted Transgenic Model?

To quickly and reliably provide overexpression of your gene of interest without worrying about positional effects like gene silencing or disruption of endogenous genes associated with random transgenesis.

General Considerations:

Targeting to the Rosa26 locus may not be amenable to tissue specific expression.

Unlike the random insertion of a transgene into the mouse genome which may be subject to position effects like silencing, variation in copy number, variable expression patterns, or the disruption of the expression of non-related endogenous genes, a targeted transgenic approach eliminates these variables by targeting a single copy of the transgene into a defined genetic locus.