Transgenic & Gene Modification, DRTC - Johns Hopkins University / University of Maryland

DRTC Baltimore Transgenic/ES Cell Core :

The aim of the Transgenic/ES Cell Core will be to accelerate research programs that depend on the development of genetically altered mouse models, including transgenics, knock-outs, knock-ins, and inducible knock-outs. The goal of the Transgenic/ES Cell core is to enable Baltimore DRTC investigators to efficiently generate genetically altered mice including transgenic, knock-out or knock-in mouse models.

The Transgenic/ES Cell Core objectives:

• Provide advice and guidance in the use of transgenic, knock-out and knock-in mice for diabetes and diabetes-related research
• Generate transgenic mouse strains
• Generate gene knock-out and knock-in mouse strains
• Provide embryo freezing and storage capabilities for Investigators

Frederic Wondisford, MD - Director
Sally Radovick, MD - Co-Director

People :

Fredric Wondisford, M.D. : Center Director and Director, Transgenic/ES Cell Core, . Phone: 410-502-5763

Sally Radovick, M.D. : Director, Pilot & Feasibility Program and Co-Director, Transgenic/ES Cell Core, . Phone: 410-955-6463

Transgenic & Gene Modification, DERC - Yale University

DERC Yale Transgenic/ES Cell Core :

The main purpose of the Transgenic Core is to provide a service facility that will ensure the ability of each DERC member to produce transgenic and chimeric mice from embryonic stem cells as well as transgenic rats. We will provide transgenic and knockout mice in the NOD system as our primary service. We will also provide transgenic mice in F2 (usually B6/C3H) and rats on Sprague-Dawley, and chimeric mice using the 129 strain ES cells. Embryo freezing will be provided as a means to preserve valuable, but not used, strains of mice. We will also establish an in vitro fetrilization procedure which will allow the cryopreservation of sperm in addition to embryos. In addition, the Core will develop a new immunodeficient rat model to be used as a system for studying human tissue grafts, BAC (bacterial artificial chromosomes) transgenic NOD mice to define specific diabetes susceptibility genes, and use NOD/129 F1 ES cells for NOD knockout and knockin experiments.

People :

Richard Flavell, Ph.D., FRS : Director, Transgenic Core, . Phone: 203-737-2216

Elizabeth E. Eynon, Ph.D. : Co-Director, Transgenic Core, . Phone: 203-785-5384

Li Wen, Ph.D. : Sub-Core Director, Transgenic Core, . Phone: 203-785-7186

Transgenic & Gene Modification, DRTC - Vanderbilt University

Transgenic / ES Cell Shared Resource :

The Transgenic / ES Cell Shared Resource assists investigators in the generation and maintenance of genetically modified mice and mouse ES cell lines. Please submit service requests to Jennifer Skelton, Managing C0Director. Additional information, service forms and policies fo ruse, can be obtained by following the link below.

Transgenic & Gene Modification, DERC - UCSD/UCLA

DERC - UCSD/UCLA Transgenic :

Core Description and Goals:

It is clear that the progress of diabetes and endocrine research during the coming decade will depend upon the ability to utilize the mouse as an experimental model to investigate both basic and clinically relevant questions in diabetes and endocrine research. This core provides members of the DERC with the most advanced technologies for genetic modification of the mouse genome. Transgenic mice carrying new or novel genes are created by microinjection of DNA into the pronuclei of fertilized eggs and "Knock-out" or “Knock-in” mice lacking specific genes of interest or containing modified version of key genes are created by homologous recombination in embryonic stem cells followed by injection into blastocysts to create chimeric mice for breeding to homozygosity. Conditional expression and Cre/LoxP targeted knock-out strategies are provided. The high degree of conservation of most sequences in genomes of humans and mice makes the approach of using transgene and knock-out gene technology to create models of human diabetes and endocrine pathogenesis extremely attractive. This Core is an outstanding example of how extraordinarily specialized techniques, highly trained dedicated personnel, and expensive equipment can be accessed by researchers who could not reasonably expect to develop them on an individual basis. The availability of this Transgenic and Knock-out Mouse Core in coordination with the Mouse Phenotyping Core, the Transcriptional Genomics Core, and Biochemistry and Molecular Assay Core, will enable our members to conduct versatile, cutting-edge, reverse genetic research in the mouse with a battery of multidisciplinary state-of-the-art techniques.

The UCSD Transgenic Mouse and Embryonic Stem Cell Core Facility has been in operation since 1992. The key measures of success of a transgenic and knock-out mouse production facility, are the measures of quality and quantity of mice produced. In 2002, we performed 71 transgenic jobs. We achieved an average of 16.35% integration for approximately 5 positive found animals per job and almost a 100% success rate overall. We also have consistent success with large DNA molecules including the injection of BACs of greater than 100 kb.

Our ES cell electroporation to produce homologous recombination to produce either knock-in or knock-out animals has also been highly successful. In 2002, we performed 121 embryonic stem cell targeting jobs resulting in an average recombination efficiency of 2.32% for an average of 4.86 homologously recombined clones per job. For ES cell injection into blastocysts for the creation of chimeric mice, we have also been very successful. We performed 90 blast injection jobs with an average of 21.9 pups per job with an average of 5 highly chimeric animals resulting for each job. These chimeras range from 30% agouti up to 100%. Mice less than 30% chimeric are not counted as chimeras. We aim to deliver at least 5 highly chimeric animals per job and thus secure an eventual germ line transmission of the targeted gene.

Services :

Pronuclear Injection
A desired DNA construct is injected into the pronucleus of a 0.5 d.p.c. Embryo. Resulting pups are returned to the Client. The client will then analyze these by Southern blot to determine the presence of the given transgene.

Blastocyst Injection
Mouse Embryonic Stem cells that have undergone homologous recombination are injected into the blastocoel cavity of 3.5 d.p.c. blastocyst stage C57Bl6 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.

Gene Targeting
A targeting vector containing a 'knock-out' or 'knock-in' gene of interest is electroporated into Mouse Embryonic Stem Cells (129 SvJ). The cells undergo drug selection, clones are picked and expanded in tissue culture, then returned to the Client for analysis. Homologous Recombinant clones are verified by the client using either PCR or Southern Blot analysis by the client.

Chromosome Count
Chromosome counting is done on targeted homologous recombinant ES cell clones prior to blastocyst injection for quality control.

Embryo Rederivation
Embryo Rederivation is used to create a pathogen free strain of mice from lines that arrive at the University from a non-approved vendor and mice contaminated with various pathogens (MHV, Pinworm, Parvovirus). Males arrive in the Rodent Isolation Unit (RIU), and wild-type females of the desired background strain are superovulated and mated to the males. Embryos are harvested in the RIU, washed and transferred into pathogen free, pseudopregnant recipient females, which are returned to the Client.

Ovary Transplants
Sexually mature females that are infertile or cannot carry a litter to term are ovariectomized. These ovaries are then transplanted into the wild-type ovariectomized females of a given background strain, and returned to the Client. Two weeks later, the recipients are mated by the client to produce transgenic pups of the original genotype.

Mouse Embryo Cryopreservation
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.

2003 Core Prices

People :

Pronuclear Injection : Jun Zhao, Core Manager , . Phone: 858-822-3270

Gene Targeting : Ella Kothari, Core Manager , . Phone: 858-534-3178

Embryo Cryopreservation : Heather Oakley, . Phone: 858-822-210

Pamela L. Mellon, Ph.D. : Associate Director of Core Support Facilities and Director, Transgenic Core, . Phone: 858-534-1312

Transgenic & Gene Modification, DERC - Boston Area

DERC - Boston Area Transgenic :

Services :

1. Generation of Transgenic Founders from DNA Constructs. Details of methodology may be found elsewhere (Hogan B, Beddington R, Constantini F, Lacy E. Manipulation of the Mouse Embryo. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1994). DNA transgenic constructs, supplied by the investigator, will be microinjected into zygotes. Tails from potentially transgenic offspring will be analyzed by the investigator to determine whether transgenic founders have been produced. If necessary, repeat injections of the construct will be performed to ensure that the investigator is supplied with at least 3 founder animals. The founder animals, their breeding and their phenotypic analysis will become the responsibility of the investigator. The cost of the DNA microinjection service will be $250 per construct for FVB mice and $1,750 per construct for C57Bl/6 mice. Please inquire about prices for other strains.

2. Cryopreservation of Mutant Mice. From time to time investigators may wish to cryopreserve embryos of valuable transgenic lines. The investigator will supply the Transgenic Core with 4 genetically modified male mice. These mice will be mated with superovulated females (of the same genetic background) and embryos will be harvested and frozen at the 8-cell stage utilizing a slow rate freezer and DMSO as the cryoprotectant. The cryopreserved embryos will be held in straws containing a maximum of 20 embryos per straw. A minimum of 600 embryos will be frozen per transgenic line. After freezing to -35 degrees centigrade, the straws will be transferred to canes and stored in liquid nitrogen cryofreezers. The embryos will be stored within the transgenic facility (current capacity is 120,000 embryos). Following freezing, 100 of the 600 embryos will be thawed and transferred to pseudopregnant recipients to assess for viability. Currently, survivability of frozen embryos in the BIDMC Transgenic Facility is ~ 90%. The cost of the freezing service will be $250 per line.

3. Microinjection of Targeted ES cell clones. The BADERC investigator will provide the transgenic facility with targeted ES cell clones. These targeted clones will be microinjected into 3.5 day old mouse embryos (blastocysts). The DERC investigator will then receive, in return, chimeric mice derived from these ES cell clones, which will then be used, by the DERC investigator, to create heterozygous and homozygous gene knockout mice. The cost of the ES cell microinjection service will be $250 per injection of two ES cell clones.

People :

Bradford Lowell, MD, PhD : Director, Transgenic Core, . Phone: 617-667-5954

Joel Lawitts, Ph.D. : Transgenic Core/Contact Person, . Phone: 617-667-7067

Transgenic & Gene Modification, DRTC - Albert Einstein College of Medicine

DRTC Albert Einstein College of Medicine Gene Modification & Transgenic Core :

The Gene Modifications Core will assist investigators in the generation of transgenic mice with modifications that are relevant to diabetes and metabolism. The Core will primarily provide services in the generation of transgenes and gene targeting constructs. Additionally, transgenic mouse colony management is occasionally an issue and the Core provides consultation regarding optimal strategies for colony production.

Services :

Transgene Construction
The Core can generate transgenes based on standard plasmids as well as large insert clones (BAC/PAC and P1 based vectors). The requesting investigator will be provided with sequence verification of the construct. For transgene constructs, the Core will provide the following services:

1. Identification of suitable promoters for universal or cell type specific expression
2. Generate chimeric plasmids containing the desired promoter and coding sequence
3. Introduce one intron and one polyadenylation signal for proper processing of the transcript
4. Design PCR based assays for identification of transgene positive mice (The requesting investigator will be responsible for generating transgenic mice.)

For BACs and P1s, recombineering is the method of choice for manipulating sequences. The Core has been successful with the use of plasmid based clones for recombineering to avoid sequence analysis of trargeted BAC clones.

1. The Core will identify and obtain the BAC clone
2. The Core will construct an intermediate plasmid containing the flanking 50bp sequences
3. The Core will construct the targeting plasmid containing the coding sequence of choice followed by an antibiotic selection marker. The Core has used both chloramphenicol and tetracycline resistance markers with success.
4. The Core will identify correctly targeted recombinant clones by PCR analysis
5. Design PCR based assays for identification of transgene positive mice

Gene targeting construction
The Core will provide the following services for generating gene targeting constructs and instruct the investigators in the following:

1. Identify the gene’s intron-exon structure from the public database
2. Identify potential alternative splicing isoforms that could interfere with the desired genetic manipulation
3. Identify introns flanking the targeted exon(s) suitable for homologous recombination in that they contain >2,000 bp of unique sequences
4. Identify one or more exons that can be flanked by loxP sites for conditional alleles or replaced for classical knockout alleles
5. Design a cloning strategy for construct generation
6. Amplification and sequence verification of the flanking sequences from genomic DNA (129 or C57BL/6) with cloning into a plasmid vector
7. Design PCR based assays that permit identification of properly targeted ES clones
8. Design PCR based assays that identify wild type and targeted alleles
9. Design PCR based assays that permit evaluation of the efficiency of CRE mediated excision of the targeted allele (The requesting investigator will be responsible for ES cell targeting and chimeric mouse production.)

Monogenic Models of Diabesity

The Animal Models Core maintains a panel of congenic mouse strains that are segregating for single gene mutations producing obesity/diabetes (Chua et al., 2002). The Core is focused on the leptin/leptin receptor system and maintains the following colonies :

• Leptin – B6-ob, FVB-ob
• Leptin receptor (mouse) – B6-db, B6-db3J, FVB-db
• GFP reporter strains – Z/EG (dual lacZ-GFP) , dsRED (dual lacZ-red fluorescent protein), Rosa26-GFP
• CRE transgenic lines – protamine CRE (general deletor strain), neuron specific CRE (Pomc-CRE, Agrp-CRE, Sf1-CRE, LEPR-B-CRE).
• LEPR transgenes in combination with db alleles (db and db3J) – Nse-LEPR and Syn-LEPR. These are useful for examining the impact of leptin on peripheral tissues and particularly relevant to issues of inflammation in obesity and diabetes.

The Core also maintains a limited number of CRE transgenic lines. Most are neuron-specific CRE lines (Pomc-CRE, Agrp-CRE, SF1-CRE,) while we also maintain a general deletor strain(Protamine CRE). These mice were generated by collaborators and are available via the Core. In general, requests for these mice are for breeding stock as further breeding with mice carrying the floxed allele of the target gene is necessary. Some of these strains were generated by external investigators and will require formal agreements to be in place prior to their use.

The Core also provides breeders from a set of congenic mouse lines that have chronic hyperglycemia but have differential susceptibility to beta cell loss and nephropathy.

Patch clamp recording and single neuron analysis
Dr. Jo will provide services related to single neuron recordings from brain slices of mice (Hinkle et al, 2004). Recent findings suggest that the hypothalamus is a major modulator of metabolism, integrating signals from circulating hormones and nutrients. However, numerous models have been proposed as suitable mechanisms. Moreover, the complexity of the interactions between various neuronal cell types and glial types is not well understood. Dr. Jo will be able to critically test the responses of known neuronal types to hormones, nutrients and neurotransmitters at the level of individual neurons. Ideally, this is done in transgenic mice with a fluorescent tag for a specific neuronal type. An example of a critical test is to determine whether an identified neuronal type responds directly to a signal or indirectly via signals from another cell type. Dr. Jo can record the response of a given neuronal type in the presence of neurotransmitter blockers and antagonists. If the responses in the presence and absence of neurotransmitter blockers are similar, then one can conclude that the responses are cell autonomous functions. However, qualitative differences in cellular responses in the presence and absence of antagonists are indicative that presynaptic influences are involved and another cell type is the primary responder. With the use of known pharmacological agents and the appropriate use of genetic models, a dissection of the molecular signaling pathway for a given signal is possible.

Dr. Jo can also perform single cell RT-PCR and fluorescent dye injections during recording sessions as an aid to characterize neurons. After recordings have identified a neuron of the desired characteristic, cytoplasm can be removed for amplification by two round RT-PCR (pioneered by Dr. J Eberwine) or fluorescent dye injected for subsequent immunohistochemical staining for a secondary antigen and visualization of projections. These methods permit the coupling of functional characteristics to neurochemical identification within the same neuron, in contrast to other methods like fluorescent cell sorting and laser capture microdissection.

Prioritization: Use of this Core is limited to Investigators engaged in diabetes-related research at Albert Einstein College of Medicine and its affiliated Institutions. In addition, the Core will consider requests from investigators at other Institutions where such services are not readily available. The Core generally supports projects that are funded by peer-reviewed grants, generally from the NIH,ADA or JDRF.

Charges: Since existing resources do not cover the total costs of services provided, the Core levies charges for its services. Charges vary depending on the assay, the volume and research being performed

Requests for Services: To request services from this laboratory, e-mail to schua@aecom.yu.edu.

People :

Streamson Coo Chua, Jr., M.D., Ph.D. : Director, Transgenic & Gene Modification Core, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Forchheimer Building, Room 511, Bronx, NY 10461. Phone: (718) 430-2986

Transgenic & Gene Modification, DERC - University of Pennsylvania

DERC - U Pennsylvania Transgenic and Chimeric Mouse Core :

The Transgenic and Chimeric Mouse Core has generated a wide range of transgenic and gene-targeted mouse lines that have proven to be powerful tools for the study of the molecular biology and physiology of Type I and Type II diabetes, obesity, and developmental and mechanistic bases for a wide variety of other disorders of endocrinology and metabolism.

A primary function of the core is to provide a centralized laboratory that will generate infection-free strains of mice carrying transgenes or targeted alterations of specific interest to individual projects in the DERC. The centralization of these technically demanding procedures in the Core laboratory results in enhanced efficiency and significant cost reduction for the DERC community.

For more information, please click here

Services :

At the present time, the TCMF provides the following services:

• DNA Injection
  

Injection of DNA into mouse eggs for the production of transgenic mice or embryos.

• ES cell Injection

Injection of embryonic stem cells into blastocysts for the production of chimeric mice.

• Embryo Cryopreservation
  

Controlled freezing of mouse embryos for preservation in liquid nitrogen.

• Mouse Line Rederivation

Surgical transfer of embryos into pathogen-free recipient females, where they can develop to term.

• Sperm Cryopreservation
  

An alternate procedure to preserve important mouse lines.

• In-Vitro Fertilization
  

Through this process, a mouse line that undergoes reproductive obstacles can be rescued.

People :

Nancy E. Cooke, M.D. : Co-Director, Transgenic and Chimeric Mouse Core , . Phone: 215-898-4425

Jean Richa, Ph.D. : Technical Director, Transgenic and Chimeric Mouse Core , . Phone: 215/898-6064 (office) or 215/573-3023

Pei Fu He, Ph.D. : Research Specialist, Transgenic and Chimeric Mouse Core , . Phone: 215-573-3023 or 215-746-6392

Haoren Wang : Research Specialist, Transgenic and Chimeric Mouse Core , . Phone: 215-573-3023; 215-746-6392

Stephen A. Liebhaber, M.D. : Co-Director, Transgenic and Chimeric Mouse Core, . Phone: 215-898-7834

Transgenic & Gene Modification, DRTC - Washington University

DRTC Washington University Transgenic Core :

Transgenic and ES Cell Core

Genetic manipulations and subsequent analysis of cells in culture have provided a wealth of information concerning the molecular and biochemical mechanisms that regulate divers functions on a cellular level. However, in vitro models do not recapitulate the complex cell-cell interactions that occur in vivo. Therefore it is important to examine the effects of specific gene products in the context of the whole animal. This is particularly important for the study of diabetes, a polygenic disorder that involves numerous tissues and is also affected by environmental factors. By using well-characterized transcriptional regulatory elements and molecular techniques, it is now possible to generate animals that either express foreign genes or have endogenous genes inactivated/mutated within specific cells or tissues. Although the concepts involved in generating such transgenic (Tg) or knockout/knockin (KO/KI) animals are fairly straight forward, it is impractical and too expensive for most labs to establish their own transgenic mouse facility.

The Transgenic and ES Cell Core overcomes these hurdles by offering transgenic mouse technology to all DRTC members. Services are provided through the Washington University Mouse Genetics Core, which has been successfully serving the Washington University research community for many years.

For more information on genetically altered mice, please visit the Mouse Genetics Core Website.

Services :

Production of Transgenic Mice

• Production of Transgenic Mice Transgenic animals are produced by injecting a DNA construct directly into the pronucleus of a newly fertilized mouse egg. In some eggs, the DNA will integrate into the mouse chromosomal DNA before cell division, and will be present in all cells in the mouse including the germ line. The Transgenic Animal Production service consists of injecting each construct into 300-350 eggs. Twenty to fifty mice will normally be born from this number of injected eggs. These animals are then screened for the presence of the transgene. The number of transgenic animals typically varies from two to eight, and these animals are transferred to the investigator at weaning. Weaning is performed at 21 days of age, so transgenic founders are received six weeks after the original egg injections are performed. For all constructs, the investigator must provide the transgenic construct prepared by a suitable protocol as well as primers for a sensitive polymerase chain reaction genotyping assay.

• Production of Chimeric Mice

  Injecting embryonic stem cells into the cavity of a mouse blastocyst produces chimeric mice. Chimeric mice resulting from injected blastocysts will be composed of tissue derived from the inner cell mass of the host blastocyst as well as the embryonic stem cells. It is possible for embryonic stem cells to contribute to all cells of the chimeric mouse, including the germ cells. Typically, chimeric mice are used to produce animals derived entirely from embryonic stem cells that have been manipulated in culture to contain a targeted mutation. Most commonly, chimeric mice are produced from embryonic stem cells derived from agouti 129/SV mice and host blastocysts harvested from black C57Bl/6 mice. The chimeric mouse production service consists of injecting embryonic stem cells provided by the investigator into 150-175 blastocysts, representing three days of work. Twenty to forty live mice are normally born from this number of injected blastocysts. Typically two to six mice will have skin with greater than seventy percent area of agouti coloring, indicating a good chance for embryonic stem cell contribution to the germline. These animals are transferred to the investigator at weaning. Weaning is performed at 21 days of age, so chimeric animals are received six weeks after the original blastocyst injections are performed. For all constructs, the investigator must provide embryonic stem cells proven to be pathogen free.

• Single Day Injection Service

  Additional injections may be paid for on a daily basis after one round of the standard service has been performed if needed to generate additional positive offspring.

• Animal Husbandry

  The husbandry service includes all aspects of maintaining mice, including inbred mice and mice containing induced mutations: breeding and weaning, tail biopsies, polymerase chain reaction genotyping, computer database colony tracking, and mouse monitoring. In addition, the Washington University Mouse Genetics Core offers ancillary services to husbandry, including injections, urine and blood collection, and dissections. Each experimental colony or group is independently tracked, and a weekly report of inventory and procedures is provided.

• Genotyping by Tail PCR

  Genotyping will be performed using a polymerase chain reaction assay and includes screening for induced mutations such as transgenes and "knockouts." Tissue for genotyping is derived from tail biopsies taken at 5-7 days of age, and assay results are normally obtained by two weeks of age.

• Rederivations

  The rederivation service takes mice that have been exposed to a specific pathogen and produces mice that are free of disease. Embryos derived from infected mice are transferred into recipient females free from specific pathogens, and these recipients are transferred to quarantine after implantation surgery.

• Cryopreservation

  Cryopreservation of mouse embryos is offered to "store" lines of animals not currently needed, to reduce cage costs and cage space, or to provide secure stock in case of contamination. The success of cryopreservation is strain dependent. Thus, feasibility and cost may vary for a particular strain.

• Ovarian Transplants

  Ovarian transplants are offered to maintain lines with normal ovaries but a phenotype that precludes breeding.

• In Vitro Fertilization

  In vitro fertilization is offered to aid in propagating mice that are poor breeders or have become too old to breed naturally. The price includes isolation and use of sperm from 2 males or eggs from up to 4 females. The success of in vitro fertilization is strain dependent. Thus, feasibility and cost may vary for a particular strain.

• Vasectomies

  Vasectomies are performed on any strain, and males are available for mating ten to twelve days after surgery.

People :

Jeffrey H. Miner, Ph.D. : Director, Transgenic and ES Cell Core , . Phone: 314-362-8235

Angela Genovese : Lab Contact, Transgenic and ES Cell Core , . Phone: 314-286-2761

Immunology, DRTC - Washington University

DRTC Washington University Immunology :

Immunology Core

The Immunology Core consists of two branches, a transgenic facility and a diabetic mice component.

Transgenic Component - Instructs and helps investigators in the production of transgenic mouse strains. Instructions are given for the preparation of DNA. Technicians are responsible for the injection and production of the transgenic mice. The facility is also capable of producing mice with ablation of genes by homologous recombination. (details)

Animal Component - The facility maintains a standardized colony of NOD mice which it supplies to investigators carrying out research in the immunology of insulin-dependent diabetes mellitus. The facility also maintains NOD mice on the SCID background. Such mice are used for transplantation of genetic strains, including gene null mice. Finally, the facility maintains SCID colony in the H-2d background (CH.17 SCID) which is used by investigators for islet transplantation experiments. (details)

Services :

Transgenic Component:

Services provided: The four genetic manipulations that the core provides include:

1) the creation of transgenic lines. The investigator prepares the DNA construct following advice given by the Director and the Technical Director, Michael White; the facility proceeds with the injections into the pronuclei of fertilized eggs, which are then planted into pseudopregnant females, prepared at the facility.

2) the targeted mutation of a gene by homologous recombination. The investigator produces the gene construct, which is then transfected into ES cells maintained by the Core. The facility is in charge of the injection into blastocysts and their implantation into pseudopregnant females. ES lines are derived from Bl/6 and 129 mice. An ES line derived from NOD has just been obtained from T. Watanabe at Kysuhu University; we are currently examining this cell line which will be extremely useful for investigations using the diabetogenic NOD mice. The targeted mutation approaches have now been extended to use gene constructs containing lox P sites and deleting it by expression of the Cre recombinase. This method allows us to generate genetically pure mutant mouse lines lacking the Neomycin-resistant gene used for selection of transfectants. Furthermore, this method combined with tissue specific expression of Cre gene allows us to generate mouse lines that lack the targeted gene function in a tissue-specific fashion.

3) the generation of mice using the blastocyst complementation procedure. These are particularly useful in studies on lymphocytes; ES cells are produced containing a mutated gene and are then injected into blastocysts derived from RAG-2 deficient mice, which do not generate lymphocytes.

4) cryopreservation of embryos. A protocol has been developed using a control rate freezer: we harvest morulas at day 2.5, and place 20-40 of them into Nunc cryo tubes mixing them with Gibco's Freezing media and DMSO.

In brief, the staff is responsible for the entire process described above, from the time it receives the DNA preparation to the production of the mice. The protocols for isolation of the eggs, their injection and reimplantation have been standardized and now followed for several years. The staff also helps the faculty with the analysis of the lines and the identification of the transgenic founders.

Personnel, management, facilities and space: The transgenic facility is directed by Robert Schreiber, a senior outstanding immunologist with extensive experience in cytokine research. He has overseen the operation of the facility since its inception 7 years ago. It is part of a larger facility run jointly by the Departments of Pathology and Immunology and Medicine. The technical coordinator is Michael White. He has extensive experience, having operated our facility since its inception seven years ago. He has been responsible for producing the 163 unique transgenic mouse lines and 50 targeted mutants in the facility. We also have a technician who helps in the microinjection (Kara Parton), and another responsible for the mouse work (Darren Kreamalmayer). The Genetics facility is located in the Specialized Research Animal Facility, a building of about 95.000 NSF of space recently constructed to hold inbred mice. This is a pathogen-free, barrier facility, AALAC accredited. We have a laboratory of 400NSF dedicated to the Genetics Core.

Contact Information: The transgenic facility director, Robert Schreiber, may be contacted at: phone: (314)-362-8747, fax: (314) 747-4888, and e-mail: Schreiber@immunology.wustl.edu.

Animal Component:

Personnel: The diabetic mice component is staffed by Osami Kanagawa and Katherine Frederick. The director, Dr. Kanagawa, has extensive experience in mouse genetics through his research on NOD diabetes and in T cell tolerance. Katherine Frederick has twenty years of experience in mouse breeding. You may contact Dr. Kanagawa at: email: kanagawa@pathology.wustl.edu, phone: (314) 362-8679, fax: (314) 362-4096.

List of mice available (italicized are new lines being generated):

NOD.SCID: NOD mouse lacking T and B lymphocytes due to the SCID mutation. Has been used extensively as a recipient for transfer of lymphocytes. The line was originally obtained from E. Leiter at Jackson Labs.

IFN-gamma receptor negative NOD: The line was produced here by Robert Schreiber and backcrossed to NOD for 15 generations. Has been used extensively by two of us, OK and RDS, to study role of IFN-gamma in autoimmunity. A new line of Stat-1 knockout mice is being backcrossed to NOD.

IFN-gamma negative NOD: The line was obtained from the Jackson Laboratory. Has been used in combination with IFN-gamma receptor negative mouse to study role of IFN-gamma in autoimmunity.

IL-4 negative NOD: The line was obtained from Dr. M. Kopf and backcrossed to NOD for 15 generations. Has been used to study role of IL-4 and Th2 T cells in autoimmunity.

I-Ea transgenic NOD: NOD mouse expressing class II I-E molecule was originally obtained from the Jackson Laboratory. Has been used to study the protective effect of non I-Ag7 class II MHC in NOD diabetes.

BDC 2.5 TCR transgenic NOD: There are two TCR transgenic mice that we maintain: i) the BDC 2.5 line was created by Mathis and Benoist. We have the TCR expressed in NOD. Kanagawa has recently made a specific monoclonal antibody to the TCR, to use for marking the cells and for biological studies; ii) a new TCR for a diabetogenic CD8 T cells was created and also maintained in NOD mice. The BDC TCR transgenic NOD on the SCID mutation has also been established in our mouse colony. The original TCR transgenic mice do not develop diabetes despite harboring a large number of potential pathogenic T cells due to the specific inhibition of disease development by regulatory T cells expressing endogenous TCR.

9.33 TcR transgenic NOD: NOD strain expressing islet antigen specific and class I MHC restricted TcR transgene. This line was developed by Kanagawa in our facility. It has been used to study role of class I MHC restricted cytolytic T cells in diabetes development.

NOD.GD: NOD line expressing I-Ad class II molecule instead of diabetogenic I-Ag7. The B10.GD mice was obtained from the laboratory of Ted Hansen and backcrossed to NOD for 13 generations. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes.

B6.g7: C57BL mouse carrying H-2g7 MHC. Has been used to study role of non-MHC genes in the regulation of diabetes development. (Line was originally obtained from E. Leiter at Jackson Labs.)

BALB/g7PD: BALB/c mouse expressing mutant I-Ag7. This line was established in our transgenic core facility. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes.)

NOD g7/PD: NOD strain expressing both wild type and mutant I-Ag7 class II MHC molecules. Has been used to study protective effect of non I-Ag7 class II MHC in NOD diabetes. The line was originally obtained from H.O. McDevitt at Stanford University).

RIP-HEL B10.BR: A new series of transgenic mice expressing the well-known protein Hen Egg-White Lysozyme (HEL) under the Rat Insulin Promoter (RIP) control. The first RIP-HEL was obtained from Christopher Goodnow: it expresses about 106 molecules of a membrane-form of HEL per beta cell. The RIP-HEL is on the B10.BR, i.e. (H-2k) background. Bred to a TCR for an HEL epitope, it develops diabetes. New lines are now being generated, including one with a cytosolic form of HEL; a new genetic construct is being prepared in which the HEL gene is flanked by lox sites; this will represent an attempt to regulate the amounts of HEL expressed after addition of the Cre recombinase.

Removable RIP-SV40 TG B6: A line now being developed by the Kanagawa laboratory--B6 mice carrying RIP driven SV40 Tag flanked with two lox-P sites. This allows us to remove the oncogenic capacity of pancreatic beta cell tumor by Cre/Lox-P mediated deletion of the transgene. After deletion of the transgene, the insulinomas and the cell lines derived from them can be used for transplantation to treat diabetic mice.

People :

Robert D. Schreiber, Ph.D. : Co-Investigator; Immunology Core, . Phone: 314-362-8747

Emil R. Unanue, MD : Director, Immunology Core, Campus Box 8108. Phone: 314-362-7440

Transgenic & Gene Modification, DRTC - University of Chicago

DRTC U Chicago Transgenic Core :

The Transgenic Core of the DRTC has been in operation since 1991. The overall objective of this Core is to continue to provide high quality, timely, and cost-effective transgenic mouse services.

1. Provide advice and guidance in the use of transgenic mice for diabetes and diabetes-related research
2. Continue to provide transgenic mice through the F1 generation
3. Continue to provide feeder and embryonic stem cell lines to investigators for knock-out and knock-in projects
4. Offer embryonic stem cell transfer to mouse blastocysts, and subsequent generation of chimeric and F1 animals as a service to Investigators.
5. Continue to provide embryo freezing and storage capabilities for Investigators
6. Provide timed pregnancy capability for invesigators
7. Provide new services for investigators including, maintenance of specific targeted mutagenesis lines, purification of DNA by facility technicians for use in microinjection, and provision of NeoR mice for the production of embryonic feeders for ES Cells.

Services :

The Core laboratory offers an annual one-day course in Mouse Handling and Breeding. This course covers all phases of transgenic and ES work as well as useful information regarding analysis and handling of the transgenic mice. The Core encourages all DRTC investigators contemplating using its facilities to take the course.

Embryonic stem cell technology has revolutionized mouse genetics, enabling researchers to remove a specific gene either from the entire genome or from a particular tissue (using the Cre-Lox recombination system). Embryo freezing is required for those situations where valuable transgenic mouse lines have been developed and where investigators plan on studying these strains for extended periods of time. If sufficient numbers of embryos are frozen, rederivation of the mouse line can be attained from implantation of thawed embryos into a pseudopregnant female. Embryo rederivations are necessary to rescue valuable mouse lines from frozen embryos, or to rederive trangenic mice that have become infected or are being transferred from dirty to clean facilities into a disease-free strain.

The following services have and will be provided:

1. Advice on vector construction for transgenic projects:

   It is provided as a free service to potential users of the Core to ensure that Core use is appropriate. The Investigator is responsible for all cloning and DNA preparation procedures in his/her own laboratory. DNA preparation for microinjection will be offered as a new service in the future

2. Preparation of transgenic mice, through the F1 generation:

   If the criteria above are met, the Core Laboratory will accept the construct(s) for injection. DNAs provided to the Core Laboratory by a Friday are usually checked by agarose gel electrophoresis for concentration over the weekend, and then injected according to a first come first served priority list as long as there is no more than a two-week backlog of constructs. If an investigator wishes to have more than one construct injected, they are asked to prioritize the constructs for the injection schedule. For each transgenic construct, females are typically mated on Sunday, Tuesday, Wednesday and Thursday nights. On Monday, Wednesday, Thursday and Friday, ~fifty fertilized embryos are injected with the DNA construct and cultured. On Tuesdays, Thursdays and Fridays, these embryos are transferred to the oviduct of each pseudopregnant female. Each day, all mice in the colony are checked. Additional operations, e.g., taking mouse tail/ear samples for DNA isolation, conducting embryo freezings, rederivations, etc., are conducted as time permits during each week Pregnant mothers are monitored until birth. As soon as a female is noticeably pregnant, Investigators are notified of the anticipated birth dates, so they can be prepared to analyze their newborn mice to determine if any seriously aberrant phenotype occurs. They are also informed that, if no aberrations occur, ear or tail samples will be available as soon as 10 days after birth. Core Laboratory technicians provide them with these samples, and the user is responsible for DNA analyses and for informing the technicians as to how long to keep the mice and which ones should be bred. The technicians are responsible for breeding, sacrificing animals, and taking tail/ear biopsies at the instructions of each user. Users are expected to provide any special instructions and to conduct all analyses. The Core laboratory has the following strain available to produce transgenic animals: CD-1, C57BI6/J, C3H, C57/CBA F1, and has developed the ability to produce transgenic NOD mice. The use of NOD mice as recipients of transgenes has broad and important implications for the use of gene manipulation in this model of spontaneous diabetes. Currently, the use of outbred CD-1 or inbred (B6xSJL) F1 mice for the generation of transgenics demands at least 10 backcrosses to the NOD strain to ensure that the NOD background is essentially pure. This procedure can take years from the time the first F1 generation are produced. In the previous funding period, the Core laboratory has developed the ability to produce transgenic mice directly in the NOD background and potentially in other inbred strains. The procedure is identical to that used for outbred strains, although special care must be taken as the eggs are more fragile during the injection procedure and of course the mice are significantly more expensive. Although the number required injections is about twice that for conventional transgenics, the yield is equivalent and the germline transmission of the transgene is essentially 100% and the time and expense saved is significant.

3. Culture of feeder and embryonic stem cell lines:

   Since 1996, the DRTC Core has been providing embryonic stem cell and gene knockout technology as a service. Lab members have evaluated three different embryonic stem cell lines: D3, CCE, and R1. When cultured on primary mouse embryo fibroblasts, the R1 line gave rise to germline transmission with high frequency, and it was therefore expanded and frozen for general use. The J1 line was added to the available cell lines in 2000. It also has a demonstrated record of high frequency germline transmission. Both lines were tested extensively for microbiological contamination prior to freezing. Presently, the Core Laboratory provides potential users with a vial of frozen R1 or J1 cells, and instructions for handling, passaging and culture. The Core also provides users with instructions for embryo fibroblast derivation and for fibroblast feeder layer preparation. Finally, the Core provides instructions on electroporation of constructs and positive/negative selection.

4. Embryonic stem cell transfer to mouse blastocysts, and subsequent generation of chimeric and F1 animals:

   The Core Laboratory requires that users take responsibility to handle the cells properly, according to instruction/guidance, and to engineer and test for their knockout. Upon demonstration by Southern blot/PCR analysis that the ES cells have been successfully targeted, Investigators contact the Core Laboratory to arrange a time to provide a designated technician with targeted ES cells, which the technician then injects into prepared C57Bl6 mouse blastocysts. Injected blastocysts are then implanted in pseudopregnant females. If the cells have been properly handled, they are easily injected, and chimeric mice are readily generated. Typically, agouti (ES) represents >75% of the coat color and is a strong indication that germline transmission will be achieved. The Core Laboratory provides at least two chimeric mice with >75% coat color from at least two independently derived targeted This is usually obtained six weeks after blastocyst injection (four weeks gestation, two week to obtain coat color). The technicians will then breed male chimeric founder animals with black C57Bl6 female animals to obtain heterozygous F1 mice; the mice will be analyzed for coat color by the Core and for transmission of the targeted allele by the Investigator. Tail samples are provided to the investigators, and once transmission has been confirmed, mice will be bred to homozygosity. The Core breeds animals to the homozygote stage, and freezes heterozygote or homozygote embryos if desired. After this time, the mice are transferred from the Core laboratory to the Investigator, and he/she assumes responsibility.

5. Embryo freezing and storage:

   For embryo freezing, Investigators indicate which mouse strains they wish to have preserved by embryo freezing. A technician mates the appropriate mice, and obtains and freezes at least 200 embryos per line. 8-32 Cell Embryos are frozen in a Planar Systems Embryo Freezer and stored in Liquid N2. Approximately 1800 vials of mouse embryos are currently stored in facility.

6. Rederivation of lines from either frozen embryos or embryos obtained from non S.P.F. mice:

   For embryo freezing, Investigators indicate which mouse strains they wish to have rederived. A technician either mates the appropriate mice, or thaws and introduces the appropriate embryos into pseudopregnant females. For mouse rederivation from dirty mice, one technician is designated to obtain the embryos, and one then transfers the embryos to clean pseudopregnant females. The service is successful as lines of mice frozen over five years ago have been successfully rederived.

7. Timed-mouse pregnancies:

   The final technical service conducted by the Core Laboratory is the timed pregnancy program. A number of faculty require mouse embryos at various ages in order to conduct developmental studies. In addition, pregnancy is a crucial time in the natural history of diabetes in view of the tendency for deterioration in glucose control. The Core laboratory makes arrangements with the Investigator to generate mice of a particular strain or construct and provide users with females at the desired stage of pregnancy.

People :

Manami Hara, Ph.D. : Director, Transgenic Core, . Phone: 773-702-3727

Roy E. Weiss, M.D., Ph.D. : Co-Director, Transgenic Core, . Phone: 773-834-7555

Linda Degenstein : Technical Director, Transgenic Core, . Phone: 773-702-0688