The same prices and service guarantees apply to BAC transgenes as to smaller plasmid-based transgenes. See the description of our Transgenic Mouse Development service described above.
PRODUCTION OF CHIMERIC MICE BY INJECTION OF TARGETED ES CELLS INTO BLASTOCYSTS
ES cell injection service: The client must supply at least one vial of frozen cells per clone. The TMF will thaw the vial, culture the cells through one passage, harvest blastocysts from C57BL/6J mice, and inject 10-15 ES cells into each viable blastocyst. Injected blastocysts will then be implanted into pseudopregnant females. The TMF guarantees that at least 40 blastocysts will be successfully injected and implanted into foster moms, or 3 chimeric pups will be produced, whichever comes first.
Testing ES cells for pathogens: ES cells supplied by the client or outside source (i.e., not produced by the TMF itself) must be tested for mycoplasma and mouse pathogens before injection. Clients must submit one extra vial of frozen cells for this purpose. Testing fee: $231/clone.
Expansion of ES cell clones to freeze back extra vials: This service is intended for cases where the client has obtained a single vial of cells, e.g., from KOMP or EUCOMM, and needs some back-up vials frozen down. The price varies according to the type of ES cells. Generally, 6-8 vials are frozen back, depending on the growth characteristics.
Expand one vial to freeze back (feeder-independent cells)$544
Expand one vial to freeze back (feeder-dependent cells) $649
(Note: This service is not currently offered to outside clients.) Cryopreservation of mouse embryos: Using both male and female mice supplied by the client, 8-cell stage embryos, harvested from superovulated and mated females, will be frozen and stored in liquid nitrogen in the TMF. The price is for one cryopreservation session, regardless of embryo yield. If sufficient embryos are obtained, the TMF will thaw a small portion and transfer them to foster moms to demonstrate ability to recover live mice. One session can accommodate up to 30 females. We require at least 15 females per strain.
Embryonic stem cell culture
ES cell electroporation, clone picking, and DNA extraction: Using a targeting construct supplied by the client, we will electroporate the construct into embryonic stem (ES) cells, grow the cells under selection, pick up to 384 colonies, split the colonies into duplicate sets of plates, freeze one set, and extract DNA from the other set. We strongly urge clients to consult with us regarding the design of their targeting constructs and alternative methods of obtaining targeted ES cells.
Electroporation of feeder-independent ES cells $5820
Electroporation of feeder-dependent ES cells $7986
Expansion of selected ES cell colonies from 96-well plates: We will thaw colonies designated by the client, expand them, freeze down ~6-8 vials of cells from each colony, and extract at least 50 ug of DNA from each colony for further analysis of the targeted allele. Prices depend on the number of clones being expanded at one time. See our website for more details.
Chromosome counting: Cells are grown, metaphase spreads prepared, and digital images of spreads are analyzed to determine the number of chromosomes per spread, for a minimum of 20 spreads. Price per clone $364
Secondary electroporation of targeted ES cell clones: We will perform a second electroporation on one targeted ES cell clone, to introduce an expression plasmid or similar construct. This service is intended for the transient introduction of a recombinase (Cre or Flp) to effect recombination of loxP or frt sites. Up to 192 colonies will be picked, split, frozen, and DNA extracted.
Secondary electroporation of one feeder-independent ES cell clone $3360
Secondary electroporation of one feeder-dependent ES cell clone $5117
Targeted transgenesis: Transgene constructs with ROSA26 homology arms are electroporated into ES cells. The cells are grown under selection and at least 24 colonies picked and split into duplicate plates. One plate is frozen and DNA is extracted from the other plate. The price includes Southern blot analysis of all picked colonies with a ROSA26 probe.
Electroporation to insert transgenes into ROSA26 locus $3360
With the completion of the genome sequence, that offers us a variety of new genes to study, and advances in combinatorial chemistry, that delivers large numbers of synthetic compounds to test, high−throughput screening (HTS) is becoming a necessary research endeavor.
We specialize in HTS of G protein−coupled receptors (GPCRs). Ligand recognition is quantified in binding experiments or monitored by second messenger responses.
One technology that we use is the FLIPR technology that allows for real−time fluorescent measurements.
Polymerase chain reaction
PCR genotyping service: The TMF will perform PCR, using mouse genomic DNA, primers, and reaction conditions supplied by the investigator. The products will be separated by gel electrophoresis and photographed to document results. Each procedure can accommodate up to 24 DNA samples.
In vitro fertilization: Using mice supplied by the client, the TMF will harvest oocytes from superovulated females and fertilize them in vitro with either fresh or frozen sperm. Embryos are cultured overnight to the 2-cell stage and implanted into pseudopregnant foster moms. If no pups are obtained, the TMF will repeat the procedure once at no extra charge. Each service can accommodate up to 12 females.
We offer two levels of Southern blotting service. The small-scale service, priced at $960 per blot, accommodates up to 24 samples and is intended for probe tests and screening of small numbers of ES cell clones (e.g., to verify correct targeting after positive clones have been identified in a large-scale screen and expanded).
(Note: this service is not currently offered to off-campus clients.) Cryopreservation of mouse sperm: For each strain, the client must supply 2 male mice (we prefer proven breeders at 3-8 months of age). The TMF will collect sperm from the epididymis and vas deferens into cryoprotective media, aliquot the sperm into 20 semen straws, and freeze them in liquid nitrogen. At least one extra straw will be frozen and thawed to test the sperm in an IVF assay, using oocytes from the same strain. Any strain producing fewer than 20% fertilized oocytes will be tested further by implanting fertilized embryos into foster moms. If live pups of the desired genotype are recovered we will consider the strain to be successfully preserved. Otherwise, we will repeat the freezing and IVF at no additional charge. Cost includes long-term storage in TMF liquid nitrogen tanks.
Tandem mass spectrometry
Understanding the broad picture in gene expression requires proteomic analyses.
The Q−TOF 2 hybrid mass spectrometer and datasystem (MS/MS/DS) is of help in these analyses.
The instrument is equipped with a database searching module for both proteins (BioLynx) and peptides (Proteinlynx) for manual and automatic searching of acquired mass spec data against sequence databases (DNA & Protein) in order to identify the unknown samples.
DNA isolation and genotyping service: The TMF will collect tail biopsies from mouse pups and ear-punch the pups for identification. DNA will be extracted from the biopsies and analyzed by PCR, using primers and a protocol of demonstrated utility, supplied by the investigator. Each procedure can accommodate up to 24 biopsies. This procedure is primarily intended for transgenic mice being bred by the TMF for an investigator.
Transgenic mouse development
Standard DNA microinjection service: DNA supplied by the investigator will be injected into fertilized eggs until at least 50 pups (or embryos) or at least 3 transgenic offspring are produced, whichever comes first. Additionally, the TMF will tail-cut all offspring, identify them by ear-punch, and deliver the tail samples to the client for genotyping (via FedEx on dry ice for off-campus clients). Transgenic pups identified by the client are shipped or transferred after weaning. Transgenic status refers only to DNA integration. The TMF cannot guarantee expression of RNA or protein from the integrated DNA. Instructions on preparing the DNA are available. The final purification step will be done by the TMF for plasmid-based transgenes. BACs must be purified entirely by the client. Brief descriptions of the construct and its expected phenotype are required.
One-Day DNA microinjection service: This service is only offered for B6SJLF1/J and FVB/N donors. If no transgenic founders are produced and the egg yield is more than one standard deviation below the average (based on the last 12 months), we will repeat the injection at no additional cost. Otherwise, the same conditions apply, as for our Standard service.