Molecular Genetics Core Mission Statement

The Molecular Genetics Service Core assists investigators with routine molecular biology applications and all technologies associated with the production and maintenance of genetically altered mouse strains. By centralizing labor-intensive tasks and providing easy access to gene targeting at vastly reduced costs we remove the burden of setting up such technologies in individual laboratories. 

Eligibility

The services are available to all members of the Department of Ophthalmology and Visual Sciences, including adjunct faculty with primary appoints in other Departments. Anyone holding an NEI grant (regardless of departmental affiliation) is also eligible to use our services. 

Description of Services

Below is our list of current services

1. General cloning and consulation

Design and construction of constructs for transgenic mice, BAC transgenics, Knock-In/Knock-Out mice, and single-guide RNAs (sgRNA) for CrispR/Cas9-mediated genomic engineering.

2. Production of Transgenic Mice

Transgenic animals are produced by injecting a DNA construct directly into the pronucleus of a newly fertilized mouse embryo. Plasmid or BAC constructs can be used. We use C57BL/6J inbred mice for these injections however; we can accommodate other strains (e.g. F1 hybrids) by special request. 

We charge per injection day and we typically do 4 injections.  The pups are screened for the presence of the transgene by a polymerase chain reaction genotyping assay. When the founder animals are at weaning age they are transferred to the investigator.   Transgenic founders are normally received seven to eight weeks after the original injections are performed when the mice are four to five weeks old.  

3. Production of Chimeric Mice

The production of chimeric mice by introducing Embryonic Stem (ES)* cells into early embryos is an alternative approach to generating transgenic mice. The technique utilizes the tissue culture system to modify and select ES cells that had received an exogenous DNA of interest. Once derived and characterized, ES cell clones may be injected into 4-day-old mouse embryos where they can differentiate into any adult tissue type.  The chimeric mouse production service consists of injecting embryonic stem cells provided by the investigator or the Washington University Mouse ES core (https://mescc.wustl.edu/) into blastocysts harvested from C57BL/6J inbred mice.  We charge per injection day and we typically do 4 injections.  The skin color of the mice from which the host blastocysts are derived is different from that of the strain used to produce the embryonic stem cells.  Mice that have skin and hair with greater than fifty percent ES cell contribution indicate a good chance for embryonic stem cell contribution to the germline. When the chimeric animals are at weaning age they are transferred to the investigator.  Chimeric animals are typically received seven to eight weeks after the original blastocyst injections are performed when the mice are five to six weeks of age.  

*ES cell derived from the major ES consortiums such as the Knockout Mouse Project (KOMP) and the European Conditional Mutagenesis Program (EUCOMM) were derived from the C57BL/6N mouse line (derived from the NIH stain of C57BL/6) and harbor the rd8 mutation. This mutation causes a retinal phenotype in many commercially available C57BL/6 mice and confounds retinal phenotypes in many genetically altered strains.  Gene targeted mice obtained from commercial vendors (even JAX) that were donated by investigators can also harbor this mutation. Investigators are encouraged to screen all mice in their colonies for this mutation. The Molecular Biology core can provide assistance. For more information see Mattapallil et al. IOVS 53:2921, 2012 and Chang et al. IOVS 54:4974, 2013.

4. Rederivation

Mice that have been exposed to pathogens or transfers of “dirty” mice from other facilities can be made free of disease through the rederivation. The infected mice are sent to Division of Comparative Medicine (DCM), they are mated in their facility and the oviducts are removed the next day by DCM personnel. The embryos are harvested and then surgically implanted into a pseudopregnant recipient in my procedure room located in SRF-W mouse facility. The pregnant females are transferred back to the DCM quarantine facility. The offspring are tested for pathogens before being released.   Normally male mice with the gene of interest are used with the advantage of ordering wild type females for mating and sacrificing for embryos for implantation.  The cost is for each rederivation day.

5. Embryo/Sperm Cryopreservation/Recovery

Freezing mouse embryos or sperm is a convenient way to preserve important mouse line for possible future use while saving animal care per diems and in receiving mouse strains of interest from other institutions.

Typically for embryo cryopreservation, seven to ten young male mice with your gene of interest will be needed to generate enough embryos for each freezing session.  The males are transferred to SRF-W animal room 111B for mating. While the males are in room 111B animal care per diems will be billed directly to the PI. Wild type females, 3 weeks of age, will be purchased by the PI and sent to room 111B. The delivery dates will be designated in a pre-planned schedule. The females are sacrificed and the embryos harvested and frozen down at the 8 cell stage.  It may take several freezing sessions to freeze down enough embryos to guarantee recovery.  It is best to have 250-350 embryos in the bank.  The cost is for each session.  A test thaw to the blastocyst stage is done each session for each strain. Frozen embryos may also be sent from other institutions for recovery in our facility. The fee is for the females used as recipients for the embryos.  Frozen embryos will be maintained in liquid nitrogen and an annual storage fee will be assessed.

Sperm cryopreservation typically requires 2 males of the strain of interest are sacrificed to harvest sperm for freezing.  The cost is for each strain. Sperm will be maintained in liquid nitrogen and an annual storage fee will be assessed.  2 males, 10-20 weeks of age that are healthy and reproductively active are typically enough to guarantee recovery. Males are brought to 710C McMillan for sperm freezing.

6. In vitro fertilization

Fresh sperm from 2 males or frozen sperm samples can be used for IVF. 20 wild type females are purchased and used for oocyte donors.

7. Other services available
  • Ovarian transplants
  • Ovariectomy
  • Vasectomy

Training can be provided for these services and fees are determined at time of services

8. Consultation and training in PCR techniques and breeding strategies

Meet the Molecular Genetics Service Core Team

Core Director

Questions and concerns should be addressed directly to Dr. Ferguson. 

Thomas Ferguson, PhD

Professor, Ophthalmology and Visual Sciences

Core Co-Director

Dr. Chen will assist Dr. Ferguson in managing the core, including reviewing projects, consultation, and addressing questions/concerns.

Shiming Chen, PhD

Professor, Ophthalmology and Visual Sciences

 

Director of the Molecular Genetics Core

 

Fee Schedule  – as of January 2018

Service

Cost

Transgenic, ES cell, CRISPR/Cas9

$ 750 per injection day

Rederivation

$ 250 per harvest day

Cryopreservation/Embryo storage

$ 200 per harvest day +Cost of liquid nitrogen

Sperm cryopreservation

$ 150.00 per harvest day +Cost of liquid nitrogen

IVF (in vitro fertilization)

$ 715

 ES cell services

Contact https://mescc.wustl.edu/ at the Washington University Mouse ES core; for services and pricing.

Fee Schedule  – As of July 2019

Service

Cost

Constructs CRISPR/Cas, transgenics, knockouts and other purposes 1,2

$ 170 per project to cover consumables

Oligonucleotides:

Billed at cost

Sequencing

$ 6 per reaction

Genotyping    

$ 2 per genotype

 Other (e.g. genotyping help) 

Negotiable

1 Oligo and/or sequencing are not included in the cost of a construct.

Transgene isolation is included in the cost of either standard or BAC transgenic constructs.