Transgenic Mouse Facility

Generation of Transgenic Mice

Service: Generation of transgenic mice by microinjection of DNA into the pronucleus of a fertilized mouse oocyte

Current Status: Since May 1997, we have generated over 730 transgenic founder mice and have averaged 10 transgenic founder mice per construct.

Service: We will purify the DNA construct insert from plasmid vectors, perform 3 days of microinjection, and provide investigators with tail biopsies so that they can identify transgenic founder mice. We will mate the founder mice to the appropriate strain and maintain these transgenic lines until the first litter is weaned. Finally, we will conduct an orientation to the investigator’s lab (if needed) about how to perform tail biopsies and how to maintain the transgenic lines.

Transgenic Mouse Time Line
(Click for full view)

Transgenic Mouse Production Request Form:
Adobe Acrobat format | MS Word format

Transgenic Mouse General Information:
Adobe Acrobat format

Transgenic/Knockout Mouse Stock Maintenance:
Adobe Acrobat format

¹Fees are subject to change without notice.

Services:

A. Generation of knockout embryonic stem cell lines that have undergone homologous recombination with the appropriate construct.

B. Microinjection of these cells into mouse blastocysts to generate knockout mice.

Current Status: Both services are fully operational. We have generated germline knockout mice from all five constructs that we have received.

Services to UTMB Investigators:

A. Generation of knockout embryonic stem cell lines: We will perform one electroporation and then pick cell lines that have survived selection (generally 300-500 per electroporation). We will cryopreserve each cell line and provide cellular extracts from each cell line for the isolation of DNA by the investigator. The investigator will analyze these DNAs by Southern blot hybridization/ PCR to identify cell lines that have integrated the construct by homologous recombination. We will then prepare these cell lines for microinjection into mouse blastocysts (i.e., amplification of the cell line). We cannot guarantee that we will generate an ES cell line that has undergone homologous recombination (since the frequency of such an event could be low, with a range generally between 1:10 to 1:500).

Knockout Mouse Time Line
(Click for full view)

B. Generation of knockout mice: We will microinject embryonic stem cell lines into mouse blastocysts and transfer the embryos into foster mothers. We will perform 4 days of microinjection for each week of microinjection requested by the investigator. Generation of a sufficient number of chimeric mice probably require at least 2-3 weeks of injection. We will mate the chimeric mice and provide the investigator tail biopsies of their offspring so that the investigator can identify mice that have transmitted the knockout allele through the germline. We will then intercross heterozygous knockout mice and maintain these lines until the first litter is weaned. Finally, we will conduct an orientation to the investigator’s lab (if needed) about how to perform tail biopsies and how to maintain the knockout lines.

Cost:
(1) Identification of knockout ES cell lines: $5400/electroporation¹.
(2) Microinjection of ES cells into mouse blastocysts: $4200/week of injection + $850 set up for the purchase of C57BL/6 male mice¹.
The cost of knockout mouse generation is subsidized by funds provided by the Sealy Center for Cancer Cell Biology, and by contributions from several departments. The cost of knockout mouse generation can vary from construct-to-construct and will depend upon: (1) whether more than one electroporation is required to generate the knockout embryonic stem cell lines and (2) the number of weeks of microinjection of the embryonic stem cell lines requested by the investigator. The investigator is responsible for the daily cage maintenance charges assessed by the Animal Resources Center (currently $0.69/cage/day), beginning with the day that microinjected blastocysts are transferred to foster mothers.

Knockout Mouse Production Request Form:
Adobe Acrobat format | MS Word format

Knockout Mouse General Information:
Adobe Acrobat format

Transgenic/Knockout Mouse Stock Maintenance:
Adobe Acrobat format

¹Fees are subject to change without notice.

Cryopreservation Services

A. Cryopreservation of Mouse embryos

Purpose: (1) To prevent the accidental loss of a valuable mouse strain as a result of natural disaster (i.e. hurricanes, floods, power loss), human error, inability to breed or infection, (2) To discontinue maintenance of a stock.

Current Status: We have cryopreserved over 4500 embryos. The viability of our control embryos as measured by in vitro culture (from the 8-cell to the blastocyst stage) is ~60%. We have performed embryo transfer of cryopreserved transgenic embryos and have recovered the expected number of mice as liveborn (~25%).

Service: For each day of cryopreservation requested, we will isolate and cryopreserve 8-cell embryos from as many as 20 superovulated female mice using the slow cooling method. We will also isolate and cryopreserve 8-cell embryos from 5 superovulated mice as controls for viability. These control embryos will be thawed and cultured in vitro to determine their viability (as measured by the number of 8-cell embryos advancing to the blastocyst stage).

Cost: The cost for this service is $450¹ per day of cryopreservation. This cost is based upon the investigator supplying the male mice needed for mating and the superovulated females (for both the strain and control mice). If we need to purchase mice, we will charge the investigator for the cost of purchasing these mice, the shipping charges, and the cost of housing the mice prior to superovulation

B. Cryopreservation of Mouse sperm

Purpose: (1) To prevent the accidental loss of a valuable mouse strain as a result of natural disaster (i.e. hurricane, flooding, power loss), human error, inability to breed, or infection (2) To discontinue maintenance of a stock.

Current Status: We have cryopreserved sperm from over 85 mice. We have successfully recovered transgenic mice from cryopreserved F1 hybrid sperm.

Service: We will cryopreserve sperm from a male mouse and distribute sperm into 8 cryovials. We will perform a sperm count and a sperm motility count of unfrozen sperm.

Cost: The cost for sperm cryopreservation is $50¹ per male mouse.

C. Recovery of cryopreserved embryos

Purpose: To recover mouse stocks from cryopreserved embryos

Current status: Fully operational

Service: We will perform embryo transfers of cryopreserved embryos into foster mothers.

Cost: Please complete the production request form and we will provide a customized quote. The investigator is responsible for the daily cage maintenance charges assessed by the Animal Resources Center (currently $0.69/cage/day), beginning with the day that embryos are transferred to foster mothers.

D. Recovery of cryopreserved sperm

Purpose: To recover mouse stocks from cryopreserved sperm.

Current status:We have successfully recovered transgenic mice from cryopreserved F1 hybrid sperm.

Service: We will superovulate female mice to obtain unfertilized eggs. We will then perform in vitro fertilization with cryopreserved sperm and culture the eggs in vitro to determine whether they advance to the 2-cell stage. Finally, we will perform embryo transfers of 2-cell fertilized eggs into foster mothers.

Cost: Please complete the production request form, and we will provide a customized quote. The investigator is responsible for the daily cage maintenance charges assessed by the Animal Resources Center (currently $0.69/cage/day), beginning with the day that embryos are transferred to foster mothers.

Cryopreservation Services Request Form:
Adobe Acrobat format | MS Word format

¹Fees are subject to change without notice.

Rederivation of Mouse Stocks

Purpose: To generate specific pathogen free mice by embryo transfer from mice that were previously infected with viruses or other pathogenic agents. In order to keep mice imported from noncommercial vendors in the Medical Research Building Animal Facility, the Animal Resources Center requires rederivation of these mice.

Current Status: Fully operational

Service: We will superovulate 5 females (per line) and then isolate the embryos from these females using sterile techniques. We will then transfer these embryos into foster mothers. The Animal Resources Center will send serum samples from the offspring for testing to verify that mice are specific pathogen free.

Cost: $675/line/session¹ plus fees (estimated to be ~$250¹) incurred by the Animal Resources Center for serology (please contact the Animal Resources Center for serology fees). The investigator is responsible for supplying the male mice needed for mating and for the daily cage maintenance charges assessed by the Animal Resources Center (currently $0.69/cage/day), beginning with the day that embryos are transferred to foster mothers.

Rederivation Request Form:
Adobe Acrobat format | MS Word forma

¹Fees are subject to change without notice.

Special Services

Investigators requesting special services should be aware that our priorities are the generation of transgenic and knockout mice. If we are not able to perform special services within a reasonable amount of time, we will let you know at the time of the service request.

1. Isolation of DNAs from founder mouse tails: We will isolate DNAs from tail biopsies of founder mice. Cost: $28.40/hour¹ + $0.20/tail¹.

2. Custom screening to identify founders: We will screen tail DNAs from founder mice by Southern blot hybridization. Cost: $28.40/hour¹ + cost of reagents (restriction endonuclease(s), chemicals, radioisotope). Please submit the service request form and we will provide an estimate of the costs.

3. Maintenance of transgenic/knockout lines: We will maintain transgenic and knockout lines generated by the facility (if they are housed in the Medical Research Building animal facility within the barrier). This service includes weaning mice, collecting tail biopsies, and setting up matings, as needed. We will provide this service on a case-by-case basis. Preference will be given to the youngest transgenic/knockout mouse strains and to collaborative projects. As space and technician time become limiting, it may be necessary to transfer the care of older transgenic mouse stocks to the PI. Cost: $28.40/hour¹.

4. Ovary Transfers
   Purpose: To maintain mouse lines that do not breed well naturally. This procedure is performed by removing the ovary from a nontransgenic mouse and replacing this ovary with the ovary from a mutant mouse. We transfer the ovaries from one mutant mouse into two recipient mice. The mice with the transplanted ovaries will then transmit the transgene to its offspring. Cost: $28.40/hour¹ + cost of mice, if needed (The estimated time of surgery per mutant mouse is ~30 minutes).

Special Services Request Form:
Adobe Acrobat format | MS Word format

¹Fees are subject to change without notice.

List of all available services:

Generation of transgenic mice
Generation of knockout embryonic stem cell lines and knockout mice
Cryopreservation services
       Cryopreservation of mouse embryos
       Cryopreservation of sperm
       Recovery of cryopreserved embryos
       Recovery of cryopreserved sperm by IVF
Rederivation of mouse stocks
Special Services
       Isolation of genomic DNA from potential founder mice
       Custom screening to identify founders
       Maintenance of transgenic/knockout lines
       Ovary transfers