Institute for Biogenesis Research
University of Hawaii John A. Burns School of Medicine

Supported by IBR-COBRE (Center of Biomedical Research Excellence) IDeA Program
NCRR 5P20RR024206-01, NIGMS 8P20GM103457-05

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Transgenic Core


University of Hawaii at Manoa Transgenic Core Facility Core
Mission Statement


The Transgenic and Embryonic Stem Cell Gene Targeting Core is a state-of-the-art facility with the expertise in the production of genetically altered subjects. Transgenic subjects carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs. Knock-out lacking specific genes of interest are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric subjects. Highly experienced personnel produce transgenic and knock-out subjects for UH investigators at very reasonable cost and with very short lead times.


Services

The Basic Services of the Transgenic Core Facility are:

  • DNA Injections
  • Embryonic Stem Cell Injection
  • Embryo Freezing

In addition to the services the Transgenic Core Facility provides, the following techniques are available to the research community:

  • Embryo Culture
  • Embryo Transfer
  • Embryo Micromanipulation
  • Mouse In Vitro Fertilization
  • Mouse Cloning Services

Pronuclear Microinjection Services

The purity of the microinjected DNA fragment is critical for successful production of transgenic founder mice. DNA impurities in the form of bacterial endotoxins or organic contaminants can decrease integration efficiency of the fragment or the viability of the microinjected embryos. The following conditions must be met by the investigator for submission of plasmid DNA:

  • A map of the plasmid, delineating the insert and relevant restriction sites.
  • Plasmid DNA is purified preferably on a cesium chloride gradient, or using of the Qiagen Plasmid Maxi kit.
  • The presence of prokaryotic sequences interferes with transgene expression. Thus the insert to be injected must be flanked by restriction site(s) that allow removal of vector sequences. The purity of the DNA preparation is to be documented with a gel photograph. The plasmid should be digested with the enzymes(s) used to release the insert from vector sequences, as well as two enzymes that cut within the insert and produce fragments of predicted size.

The Facility also notes that a transgene construct prepared from genomic DNA is preferable to one prepared from cDNA. Although there are reports of cDNA expression in the literature, the presence of introns increases the likelihood of transgene expression.

Once all of the above criteria are met, please submit a completed electronic application, DNA construct, and supporting documentation. Users will be scheduled for microinjection services on a first-come, first-serve basis. Notification of the scheduled microinjection date will be sent via e-mail within 10 working days of the receipt of all application materials.

OUR GUARANTEE

The TCF guarantees at least one founder carrying all or part of the transgene.

If there are no founders after one injection session, the PI will meet with TCF personnel to review the transgene screening strategies. A minimum of two strategies must be tested. In addition, investigators must demonstrate good genomic DNA quality by running internal controls using Southern blot analysis or PCR on all tail DNA samples. Once all these criteria are met, a second microinjection will be scheduled. If after a second injection session there are still no founders, no future microinjections will be performed. We will also review the possibility of embryonic lethality and discuss procedures to test for this outcome. If embryonic lethality is demonstrated, the PI is responsible for full payment for microinjection services and no additional microinjections will be performed.

The PI understands that while we guarantee at least one founder carrying all or part of the transgene, we do not guarantee transgene expression or germline transmission. If the transgene is embryonic lethal, the PI understands that he/she is responsible for payment in full of fees associated with microinjection, and agrees to this by signing the first page of the application.

The application and supporting documentation can be faxed to (808-956-7316), or hand delivered to the facility with 100 micrograms of high purity plasmid DNA.

Following purification and quantification of the DNA fragment, the construct will be microinjected into one-cell mouse embryos. The TCF produces transgenic mice on an B6D2F1 X B6D2F1 genetic background. Alternate backgrounds can be used, but Investigators should discuss these preferences with the TCF prior to submitting an application.

The TCF ear punches mice at postnatal day 10 and provides tail samples to the investigator. The investigator is responsible for genotyping potential founder transgenic mice and establishing the line of mice from each founder. The TCF strongly recommends screening potential founders by Southern Blot. A working genotyping strategy must be demonstrated prior to project initiation.

Cost

University of Hawaii Investigators: $2,500 *

External Academic Non-Profit Investigators: $3,500 **

Industry: Contact the TCF for pricing.

*Price excludes the purchase of female donors and per diem expenses, which are charged directly to the investigator by TCF. Contact the Core for an estimate of the additional charge.

**Price excludes the purchase of female donors and per diem expenses. The actual amount incurred by the TCF for these purchases will be added to the final bill. Contact the Core for an estimate of the additional charge.


Preparation of DNA for pronuclear microinjection

General considerations

The quality of DNA for microinjection is essential to the success of transgenic experiments. DNA that is not purified properly will make the injections difficult and/or reduce the survival. Any traces of phenol, ethanol, salts or enzymes are toxic for embryos. It is also important to get rid of any particles that could clog the injection needles. Glassware with possible detergent residue should be avoided; disposable plasticware rinsed before use with filtered water is used instead.

Prefilter all DNA suspension solutions and microinjection buffer through 0.02 um filters prior to diluting the DNA for microinjection (the 0.02 μm filters will trap DNA molecules) with Anotop syringe filter - Whatman http://www.whatman.com/ 6809-1102. The use of silicone free tubes prevents potential clogging problems.

18 Ohms Milli-Q water, sterile endotoxin-free ultra-pure water from Sigma http://www.sigma-aldrich.com/; Gibco http://invitrogen.com or embryo-tested water (e.g. Sigma W1503) should be used for microinjection buffer.

The DNA concentration is a crucial factor to the success of experiments. Excess DNA is toxic and results in developmental arrest. Too diluted DNA will decrease the number of transgenic founders. Errors in concentration can significantly affect transgenic production. We require 50 μl of DNA fragment at the concentration of 50-100 ng/μl. The problems with clogged needles during microinjections could be avoided to a large extent if the concentration of the stock DNA solution after purification is at least 50 ng/μl. Additional centrifugation is performed by TG Core, so only the upper portion of DNA solution is used for the preparation of final dilutions (at least 10-20X) immediately before microinjections.

A screening method to identify transgenic animals needs to be established before submitting service request or DNA for microinjection. To provide the evidence of PCR or Southern blot assay that detects transgene at single copy concentration, wild type tail DNA is spiked with a known amount of transgene DNA to produce copy standards. A Southern blot assay should be used to determine the copy number, integration site number, and the transgene integrity in the founders prior to breeding.

Procedure

1. Purify recombinant plasmid (e.g. Qiagen Endo-Free Plasmid Maxi Kit 12362 or CsCl gradient).

2. Digest ~50 ug of plasmid with appropriate restriction enzyme, removing as much vector sequences as possible from the insert as they could inhibit the expression of transgene and may be toxic to the zygotes. Run a minigel to check the digest. Note: If the transgene insert size is similar to the vector size, add enzyme digesting the vector

3. Separate the insert from the vector on 0.8% agarose gel run in TAE.

4. Cut out the band of interest using clean razor blade and transfer gel slice into a DNase-free Eppendorf tube minimizing DNA exposure to UV light.

5. Purify the insert by one of the following methods (recommended to repeat twice):

  • QIAquick PCR purification kit (Qiagen 28106)
  • UltraCleanTM GelSpinTM Kit (Mo Bio Laboratories 12400-250)
  • Electroelution, ethanol precipitation, ion exchange column (e.g. Elutip-D mini-column Schleicher and Schuell 462615 or Xymotech 27370)
  • GENECLEAN kit (Q.BIOgene 1001-200)
  • NucleoSpinTM Extract Kit (Clontech K3051-1)
  • QIAEX II gel extraction kit (Qiagen 20051)

It is strongly recommended using powder-free gloves when handling DNA preparation to avoid potential clogging of injection needles.

6. Resuspend recovered DNA in filtered microinjection buffer.

5-10 mM Tris pH=7.4-7.5, 0.1-0.25 mM EDTA prepared in high quality water from: 1M Tris-HCl stock (Sigma T2663) and 0.5 M EDTA stock (Sigma E7889)

e.g. for 10mM Tris-HCl pH=7.5; 0.15 mM EDTA for 10 ml: 100 ul 1M Tris-HCl pH7.5 3 ul 0.5 M EDTA

Make up one liter and run 500 ml through 0.22 uM filter unit (e.g. Sigma Catalog no. F9893). Discard the first 500 ml to wash the filter. Filter the second 500 ml through the filter and keep in the provided sterile container. Filter through a 0.02 micron Anotop syringe filter prior to diluting the DNA for microinjection (Whatman cat. no. 6809-1102).

Injection buffer is available from Millipore (MR-095-10F http://www.millipore.com/catalogue/item/mr-095-10f ) and can be provided by the TG Core

7. OPTIONAL (recommended for the constructs not larger than 5 kb). Filter DNA suspension through 0.2 or 0.45 um not-sterilized filters pre-washed with water or microinjection buffer. DO NOT use filters that have been sterilized with ethylene oxide!

  • Millipore 0.45 (UFC30HV00) or 0.2 um (UFC30GV00) ultrafree-MC filters
  • Millipore Millex HV 0.45 um (SLHVR04NL)
  • Spin-X centrifuge tube-filters 0.45 um (Costar 8162)
  • Dynagard 0.2 um Hollow fiber syringe filters (Microgon DG2M-330-50S)

8. Determine the concentration and validate DNA fragment

  • Make up an appropriate dilution and read 260/280 nm on a standard UV spectrophotometer keeping in mind that it is not accurate enough to determine the final concentration used for microinjections (1-5 ng/ul) and it gives no indication of the integrity of the DNA.
  • If possible use NanoDrop to quantify and assess the purity of the final prep. Please, refer to the NanoDrop Nucleic Acid Purity ratios (PDF);
  • Run a minigel with molecular weight markers of known concentration such as DNA mass ladder (e.g. DNA molecular weight ladders from http://www.norgenbiotek) compare staining intensities. It is best to run several volumes of diluted DNA fragment with serial dilutions of the standard. Estimate the DNA concentration of the sample. Check the purity of the DNA. Make sure that it is intact, of right size and there is no smear of sheared DNA.
  • Adjust the final concentration to the desired 50-100 ng/ul with FILTERED microinjection buffer. Wash tubes and tips with microinjection buffer or filtered water before use. The final dilution of DNA fragment will be done directly before microinjection.

9. Label the tube with construct name, concentration, and date and store at -20 C 10. Submit the picture of the gel and prepared DNA fragment to the TG Core.

10. Submit the picture of the gel and prepared DNA fragment to the TG Core.

Blastocyst Microinjection Services

Mouse Embryonic Stem cells that have undergone homologous recombination are injected into the blastocoel cavity of 3.5 d.p.c. blastocyst stage C57BL/6 Embryos. Injected blastocysts are implanted into pseudopregnant recipient females, and chimeric pups are born. Chimerics are returned to the Client. Those that transmit the 'knock-out' allele to germline produce agouti pups, which are analyzed for phenotype by the client.

Embryonic stem cell clones to be provided by the user will be microinjected into a minimum of 40 C57BL/6 blastocysts per session. Prior to injection of ES cells into blastocysts, investigators are required to:

1. Fill out an application form. The application should be downloaded, completed, and emailed.

2. Provide the TCF with ES cell clones. The ES cells should be mailed/delivered to the TCF when the investigator has been notified of a start date. The TCF recommends maintaining the storage method of cells obtained from an external source, the same as they were received.

PLEASE NOTE: ES cells from sources other than the TCF must undergo Mycoplasma (pulmonis), mouse hepatitis virus and Sendai testing. The TCF recommends sending cells to Charles River Laboratories (1-800-338-9680), as they guarantee a 10 day turnaround and charge a nominal fee. Contact TCF for more information.

The TCF will nurture chimeric mice up to 21 postnatal days, at which point animals will be transferred to the investigator, who will be responsible for breeding and establishing the line of mice. Offsite investigators will arrange the transfer with the TCF in conjunction with their animal facility.

Cost

University of Hawaii Investigators: $2,500 *

External Academic Non-Profit Investigators: $3,500 **

Industry: Contact the TCF for pricing.

*Price excludes the purchase of female donors and per diem expenses, which are charged directly to the investigator by TCF. Contact the Core for an estimate of the additional charge.

**Price excludes the purchase of female donors and per diem expenses. The actual amount incurred by the TCF for these purchases will be added to the final bill. Contact the Core for an estimate of the additional charge.

Embryo Cryopreservation Services

Genetic banking through the use of mouse embryo cryopreservation is a practical means to store scientifically valuable mice. Mouse embryos stored in liquid nitrogen offer a safe way to preserve lines with potential future use but no current use and offer savings in facility space and the expense associated with keeping a breeding colony. Another advantage is that during the process of embryo collection, most pathogenic organisms are excluded as in other methods used to rederive lines of mice.

Briefly, the procedure entails harvesting embryos from donor females at the 8-cell developmental stage that is optimal for cryopreservation by our method. A controlled-rate methanol bath freezer is used to gradually cool the embryos to -80°C, before plunging them into liquid nitrogen for storage. Quality control is assured by freezing and thawing a representative sample of wild-type frozen embryos along with each line frozen down. Frozen embryos should be retrieved by the investigator one week post-freezing for long-term storage in liquid nitrogen in the investigator's own lab.

Cost

University of Hawaii Investigators: $2,500 *

External Academic Non-Profit Investigators: $3,500 **

Industry: Contact the TCF for pricing.

*Price excludes the purchase of female donors and per diem expenses, which are charged directly to the investigator by TCF. Contact the Core for an estimate of the additional charge.

**Price excludes the purchase of female donors and per diem expenses. The actual amount incurred by the TCF for these purchases will be added to the final bill. Contact the Core for an estimate of the additional charge.

Intracytoplasmic Cell Injection (ICSI) Services

ICSI is an acronym for intracytoplasmic sperm injection - which is a fancy way of saying, "inject sperm in the middle of the egg". ICSI is a very effective method to fertilize eggs to treat male infertility problems. Artificial Fertilization by Intracytoplasmic Injection (ICSI) service is available. Please contact TCF to set up experiment and for more information.

Cost

University of Hawaii Investigators: $2,500 *

External Academic Non-Profit Investigators: $3,500 **

Industry: Contact the TCF for pricing.

*Price excludes the purchase of female donors and per diem expenses, which are charged directly to the investigator by TCF. Contact the Core for an estimate of the additional charge.

**Price excludes the purchase of female donors and per diem expenses. The actual amount incurred by the TCF for these purchases will be added to the final bill. Contact the Core for an estimate of the additional charge.

Contact Us

Facility Director and Lab Manager: Joel S. Marh

Phone: (808) 956-7064

Fax: (808) 956-7316

Email: marhj@hawaii.edu

Office: Institute for Biogenesis Research Building, Rm. E-106

Mailing Address: Institute for Biogenesis Research

John A. Burns School of Medicine
University of Hawaii at Manoa
1960 East-West Rd.
Honolulu, HI 96822

Molecular Specialist: Zoia Stoytcheva
Phone: (808) 956-9684

Fax: (808) 956-7316

Email: zoia@hawaii.edu

Office: Institute for Biogenesis Research Building, Rm. E-109

Mailing Address: Institute for Biogenesis Research
John A. Burns School of Medicine
University of Hawaii at Manoa
1960 East-West Rd.
Honolulu, HI 96822