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Mouse Resource Facility

The Mouse Resource Facility (MRF) was added to the GEMF in 1999 as a means to supply the need for mouse reagents. The MRF stocks numerous mouse reagents. The MRF also provides northern blots from adult tissues and staged embryos. It provides genomic DNA, cDNA libraries, BAC filter sets and vectors for the generation of targeting constructs. Additionally, the MRF maintains a small colony of Cre and lacZ transgenic mice essential for the generation of conditional deletions in mice. These reagents are generally provided free, or for a small fee to cover maintenance costs.

BAC (Bacterial Artificial Chromosome) Libraries

Once a gene has been identified and partial sequence is obtained, it is easy to locate the gene using the mouse genome sequence databases. This location can then be used to identify a BAC clone containing the gene, from which the gene itself can be cloned, sequenced and manipulated to produce a construct for testing in transgenic or gene knock-out/in mice.

We have two BAC libraries that we provide for investigator use. Each is composed of high-density filters containing DNA derived from colonies representing either a C57BL/6J mouse library or a 129 SJ/SvEv mouse library. Both libraries were obtained from BAC/PAC Resources at the Children's Hospital Oakland Research Institute (CHORI). Each hybridization membrane represents over 18,000 distinct mouse BAC clones, stamped in duplicate. Individual clones identified after screening can be obtained as stab cultures at a cost of $20 per clone in addition to a $35 set-up charge per shipment from CHORI.

  • RPCI-22: 129S6/SvEvTac Mouse BAC Library
  • RPCI-23: C57BL/6J Mouse BAC Library

Cost: $30/use for MD Anderson investigators; $60/use for external investigators

Northern Blot Filters

To determine the spatial and temporal pattern of expression of a gene, a northern blot analysis is generally performed. We are attempting to make this analysis easier for the investigator by providing RNA from adult tissues and staged embryos that is immobilized onto filters and ready for probing to detect expression of the identified gene.

We have two northern blot filters that we provide for investigator use. One is of total RNA isolated from mouse adult tissues and the other is total RNA from mouse embryos at stages from e4.5 through e18.5. Both filters were obtained from Seegene, Inc.

  • MATB R1015: Mouse Adult Tissue Blot
  • MESB R1011: Mouse Embryo Full Stage Blot

Cost: $30/use for MD Anderson investigators; $60/use for external investigators

cDNA Libraries

To construct a vector using a large genomic fragment is often unwieldy and technically difficult. For that purpose, we are providing cDNA libraries so that the shorter cDNA clone may be obtained.

We will provide the investigator with an aliquot of a cDNA library prepared from 11.5 day mouse embryos. This Clontech library was amplified in XL1-Blue and utilizes lambdaTriplEx as the cloning vector. The average cDNA size is 1.9 kb, and ranges from 0.5-3.8 kb. Three million independent clones are represented, and the titer is > 108 pfu/ml.

Cost: $30/aliquot for MD Anderson investigators; $60/aliquot for external investigators

Plasmids

The MRF stocks a variety of plasmids commonly used in the construction of DNA for the manipulation of the mouse genome. These include plasmids containing the neomycin gene that confers G418 resistance, Cre-loxP containing plasmids, plasmids containing the FRT sites used by FLP recombinase, LacZ containing plasmids and various other useful plasmids.

The following plasmids may be obtained by the investigator for use in generating targeting constructs. Five-microliter (5-µl) aliquots of the plasmid of choice will be supplied to the investigator for bacterial transformation.

  • PGK-Neo-bpA loxA

Contains the neomycin resistance gene expressed from the PGK promoter with the bovine growth hormone poly A site and flanked by lox P sites for easy removal. The vector used is pBluescript II KS (-). Directionality is from the T3 to T7 promoter (A).

  • PGK-Neo-bpA loxB

Contains the neomycin resistance gene expressed from the PGK promoter with the bovine growth hormone poly A site and flanked by lox P sites for easy removal. The vector used is pBluescript II KS (-). Directionality is from the T7 to T3 promoter (B).

  • pMC1TKbpA

Contains a TK cassette flanked by polycloning sites. Obtained from Allen Bradley.

  • PGK-Neo-bpA FRT

Contains the neomycin resistance gene expressed from the PGK promoter with the bovine growth hormone poly A site and flanked by frt sites for removal with flp recombinase.

  • IRES-Cre-pA FRT2 PGK-Neo-bpA

Contains a 2.4-kb XhoI/SmaI fragment with an IRES-Cre-pA gene obtained from plasmid IRES-Cre-pA, and a 2.5-kb EcoRV/BamHI fragment from plasmid FRT-PGK-Neo-bpA FRT-6 in pBluescript II KS (-). The neo gene is flanked by FRT sites for removal with flp recombinase.

  • PGK-hygro

Contains the hygromycin-resistance gene expressed from the PGK promoter. The gene is flanked by multicloning sites.

  • IRES-Cre-pA

Contains a ~1.0-kb XhoI/BamHI fragment with the IRES-Cre and a ~1.4-kb BamHI/SalI fragment with the polyA site for Cre from plasmid pOG231 all in pBluescript II KS (-).

  • pIRES-T-lacZ/loxP-Neo

Contains a tau-LacZ gene which can be expressed from an IRES as well as a loxP-flanked neo gene expressed from the PGK promoter. Both the LacZ gene and the neo gene are flanked by polycloning sites.

  • pPGK-puro-bpA-loxP

Contains the puromycin-resistance gene expressed from the PGK promoter with the bovine growth hormone polyA site and flanked by lox P sites for easy removal.

  • pOG231

A plasmid containing the Cre gene expressed from the CMV promoter for universal expression. Obtained from Steve O'Gorman.

  • pCAGG-Flpe

A plasmid containing the flp-recombinase expressed from the CMV promoter that was manipulated to conform to the Kozak consensus sequence for higher levels of expression. Obtained from Francis Stewart.

Cost: There is no charge for plasmids

Mouse Resources

The MRF maintains a small number of Cre, GFP, p53 and LacZ-containing mice, and will distribute these to MD Anderson researchers (only) upon request. To determine what mice are available, please contact Jan Parker-Thornburg. 

Mice cannot be sent to external investigators. However, the GEMF will be happy to supply ordering information to external investigators. 

  • CMV-Cre mice

Mice transgenic for the CMV-Cre gene. Cre is expressed in all cells constitutively. These mice are in a C57BL/6 background.

  • R26R mice

Mice used for reporting Cre expression. A ROSA26-loxP-Neo-loxP-LacZ gene was targeted to the ROSA26 locus (Soriano P. [1999] Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genetics 21, 70-71.) Constitutive, high level expression of the lacZ gene is seen in all cells if Cre recombination removes the Neo resistance gene. These mice are in a C57BL/6:129 hybrid background.

  • Zp3-Cre mice

C57BL/6 transgenic mice containing the Cre recombinase gene expressed from the Zp3 promoter. "Cre is expressed exclusively in the growing oocyte prior to completion of the first meiotic division." (Lewandoski M, Montzka Wassarman K, Martin GR [1997] Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Current Biology 7, 148-151.)

  • FLPeR mice

A deletor strain of mice used for generalized high level Flp recombinase expression. The ROSA26- gene was targeted with the enhanced version of the site-specific recombinase FLP (Farley FW, Sorioano P, Steffen LS, Dymecki SM [2000]. Widespread recombinase expression using FLPeR (flipper) mice. genesis 28, 106-110.) Constitutive, high level expression of FLPe is seen in all cells. These mice are in a 129 background.

  • Autosomal GFP

Mice that carry the green fluorescent protein driven by the chicken beta-actin promoter and the CMV intermediate early enhancer. "This strain can be used as a source of fluorescently marked cells or tissues. The transgene is expressed in all nucleated embryonic tissues...Though ubiquitous, expression levels vary between different organs" (The Jackson Laboratory Web site: Strain 003115). The reference for this strain is Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A. (1998). Generating green fluorescent mice by germline transmission of green fluorescent ES cells. Mech. Dev. 76, 79-90.

Cost: There is a $30 charge per mouse. For excess stock animals (non-transgenic, retired breeders) there is a charge of $15 per mouse.

Expansion of a Vial of ES Cells

  • This includes expansion of ES cells brought in from outside (e.g.:  KOMP or EUCOMM cells) and cytogenetic testing of these cells. Only those cells that are >60% diploid are eligible for injection
  • Fee: $400/sample ($300 for expansion and $100 for testing) for MD Anderson investigators; $800/sample ($600 for expansion and $200 for testing) for external investigators
  • Available at both North and South Campus sites
  • THIS SERVICE IS INCLUDED IN THE KOMP/EUCOMM SERVICE REQUEST FORM

Supplies

The GEMF can supply small amounts of chemicals for ES cell culture, embryo shipping and superovulation. We can also supply morulae for culture to blastocyst for both C57BL/6 and BL/6 albino strains.

Training

The GEMF can train limited numbers of people in pronuclear injection and ES cell culture. This will typically be a one-on-one training, and will require a substantial time commitment from the trainee. To avoid cross contamination, trainees cannot work with their own mice prior to coming in for daily training. 

Contact

To obtain reagents, please contact Jan Parker-Thornburg by phone at 713-745-2654, or by e-mail at jpthorn@mdanderson.org.


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