• Genome Edited Mice and Rats using CRISPR/Cas9
• Custom Transgenic Mouse Models: 3 transgenic founders are guaranteed

Routinely prepared in (C57BL/6 X SJL)F2 mice. For other genetic backgrounds contact [email protected].

• Custom Transgenic RAT models: 3 transgenic founders are guaranteed

Routinely prepared in Sprague Dawley rats – Crl:CD (SD). Other genetic backgrounds are available – contact [email protected]. 

Bacterial Artificial Chromosome Recombinering Core

1. purify BAC DNA for transgenic mouse or rat production
2. restriction map BACs on pulsed field gels
3. use recombineering to genetically modify BACs

Gene Targeting Service: We work with you to “knockout” a gene in a mouse.

1. Electroporation of 129 mouse or C57BL/6 mouse derived embryonic stem cells.
2. Expansion and chromosome counting of ES cell clones.
3. ES Cell Injection: 60 blastocysts are guaranteed to be injected per ES clone.

Personnel Training

1. microinjection of DNA or ES cells and production of genetically altered mice
2. ES cell culture and manipulation

• Plasmids and certified ES cell culture reagents
• Centralized Support for transgenic animal production
• Conversion of mouse lines to specific pathogen free statu
• Embryo cryopreservation and recovery

Transgenic Mouse Production

• Three transgenic mice are guaranteed.
• Mice are produced in the (C57BL/6 X SJL)F2 genetic background.
• Other genetic backgrounds may be specified by investigators (additional cost).
• Large DNAs (BACs, YACs, P1s) may be specified at no additional cost.

Transgenic Rat Production

• Three transgenic rats are guaranteed.
• Rats are produced in the Sprague-Dawley genetic background  (Crl:CD (SD)IGS BR).
• Other genetic backgrounds may be specified by investigators (additional cost).
• Large DNAs (BACs, YACs, P1s) may be specified at no additional cost.

Transgenic Mouse & Transgenic Rat Outline

Transgenic rats are available from the University of Michigan Transgenic Core.

The Transgenic Facility routinely produces transgenic rats for biomedical research. We have developed considerable expertise in this area (Filipiak and Saunders, 2006). The general outline of producing transgenic rats and transgenic mice is the same.

The efficiency of transgenic rat production is equivalent to the production of transgenic mice in inbred mouse strains such as C57BL/6J.

We have generate transgenic rats in the following genetic backgrounds:

• Fischer 344 Rats – F344/NHsd
• Long Evans Rats – Crl:LE
• Sprague Dawley Rats – Crl:CD (SD)
• Wistar Rats – Crl:Wi

We have successfully generated:

• CRISPR/Cas9 gene knockout and gene knockin (Cre, Flpo, etc.) rat models
• Plasmid transgene transgenic rats
• Bacterial artificial chromosome transgenic rats
• Knockout rats by the pronuclear microinjection of zinc finger nuclease mRNA.

If you are interested in obtaining transgenic rats from the Transgenic Facility, please contact [email protected] for more information.

Purpose: to produce embryonic stem (ES) cells with mutations induced by homologous recombination with a targeting vector.

Phase I:

The investigator demonstrates a sensitive screen for homologous recombination and clones the targeting vector. Core staff electroporate ES cells, isolate clones, and prepare DNA from clones.

Phase II:

The investigator screens ES cell DNA and identifies recombinants. Core staff expand ES clones from frozen stocks, count chromosomes, and the investigator confirms gene targeting in thawed ES clones.

Phase III:

The investigator and Core staff select ES cell clones for recombination. Core staff microinject ES cells into C57BL/6 blastocysts to produce chimeras.

Phase IV:

The investigator breeds the chimeras to obtain germline transmission and analyzes the resulting animals.

Gene Targeting Outline

• Microinjection of embryonic stem (ES) cells into C57BL/6 blastocysts.
• Microinjection of C57BL/6 derived ES cells into albino C57BL/6 blastoycsts.
• We guarantee to inject 60 blastocysts with each ES cell clone.
• The production of germline chimeras depends on the quality of the ES cells.

• Preparation of ES cell lines from mouse blastocysts.
• We will collect blastocysts from your genetically engineered mouse model and culture them under conditions that are conducive to establishing ES cell lines.
• The number of ES cell lines produced is propotional to the number blastoycsts available for culture.
• The success of the Transgenic Core in de novo ES cell line derivation meets or exceeds published standards.

Service Description

• Pluripotent ES cell lines tested for germline chimera formation (see list of ES cell lines).
• Fetal bovine serum tested for ES cell proliferation with minimal differentiation.
• Feeder cells (neor) for ES cell culture (5X106 cells per vial).
• Plasmids for gene targeting, Cre/loxP technology, FLP/FRT technology, reporters, etc. are available.
• Genomic lambda library prepared from 129X1/SvJ mice.
• Genomic DNA isolated from ES cells in 96-well plate format so that you can test your screening method.

Access to cell lines and plasmids is only provided to investigators who have complete, approved material transfer agreements.

CRISPR/Cas9 Overview

CRISPR/Cas9 use RNA guided engineered nucleases (RGEN) to produce double strand breaks in a chromosome. In order to divide and grow cells must repair the break. Two cellular mechanisms used for this purpose are non-homologous endjoining (NHEJ) and homology directed repair (HDR). When NHEJ repair occurs small deletions and insertions may occur on the chromosome. If these events occur in a critical exon then protein expression will be disrupted and the gene will be "knocked out." If the cell copies new DNA sequence from a experimental DNA template during HDR then the cell will have a new DNA sequence and can express an altered protein or an exogenous protein such as a fluorescent reporter. If the cell fails to repair the damage then it will fail divide and its daughter cells will be absent from the cell culture.

The microinjection of CRISPR/Cas9 plasmid DNA, mixtures of guide RNA (sgRNA) and Cas9 nuclease mRNA, or mixtures of Cas9 protein and sgRNA into fertilized mouse or rat eggs is a specialized application that differs from cell culture experiments in several ways. The transfection efficiency is 100%, reagents are physically introduced in each egg, one by one. The number of cells that are transfected in a single experiment much lower than can be achieved in cell culture. The absence of daughter cells that do not repair chromosome breaks is manifested as the absence of live mice and rats when pups are born. The pups that are born are euploid and do not carry multiple copies of chromosomes, as is common in long-established cell lines used in routine cell culture work.

The use of the wild type Cas9 enzyme with two functional nucleolytic domains in combination with sgRNA targets that are only 20 bp long or less has drawn interest to the question of off-target cleavage events (Gabriel et al., 2015). In animal models, the backcrossing of founders carrying Cas9-induced mutations to wild type animals will result in the segregation of off-target mutations from the genetic change of interest. In cell culture models this approach is not possible. In order to address concerns raised by off-target hits the use of Cas9 nickase (Cas9n) that carries a mutation from aspartic acid to alanine at residue 10 has been employed. In our hands, there is evidence that the Cas9n has lower activity than Cas9, however the specificity of cleavage is higher and the presence of off-targets hits is near the limit of detection.

The Transgenic Core has microinjects Cas9 plasmid DNA to produce gene knockouts and knockins based on the pX330 plasmid (pX330-U6-Chimeric_BB-CBh-hSpCas9, Ran et al., 2013). This plasmid is available from Addgene.org (Plasmid #42230). The Transgenic Core has used plasmid used to produce gene disruptions, oligonucleotide edited genes, reporter knockins and conditional (floxed) genes in mice. In rats, gene disruptions and oligonucleotide knockins have been produced with several reporter knockins in progress. When concerns regarding off targets are a concern, as in cell lines, the Transgenic Core uses a modified plasmid (pX330A_D10A-1x2) that simultaneously expresses two sgRNA and the Cas9 nickase to introduce specific chromosome breaks (Sakuma et al, 2014). This system can be used to co-express as many as seven sgRNA along with either the Cas9n or Cas9 protein (plasmids are available from Addgene.org).

sgRNA Algorithms

The choice of an sgRNA targeting a critical exon for a knockout or a non-coding region for a gene knockin is key to success. For this purpose many algorithms have been published and are available through the internet. A partial list includes the sites listed below. The Transgenic Core prefers to use the algorithm published by Haeussler et al. (2016) because the algorithm ranks sgRNA sequences according to a prediction of activity when delivered from a U6 promoter in a plasmid or when delivered as an RNA molecule.

• CRISPOR
• Broad Institute Design sgRNAs for CRISPRko
• ChopChop (Harvard)
• WTSI Genome Editing (WGE)
• dkfz. E-CRISP
• COSMID: CRISPR (Georgia Tech)
• DBCLS CRISPR Direct
• MIT CRISPR Design

After sgRNA targets are selected for mouse or rat genes, we clone the targets into a pX330 based plasmid and test it for activity by electroporation of mouse embryonic stem cells, rat fibroblasts or microinjection into fertilized eggs. For an example of the results from one of these tests see Activity of CRISPR/Cas9.

To produce mouse and rat knockouts and knockins we microinject circular plasmid DNA into fertilized mouse or rat eggs. If desired, oligonucleotides or DNA plasmids are co-injected with the Cas9 or Cas9n plasmid to introduce knockins. When designing the HDR templates it is important to modify the donor DNA sequence so that it will not be a substrate for Cas9 or Cas9n cleavage. The length of the homology regions that match to the chromosome vary according to the type of knockin desired and the sequence in the target locus. It should be noted that the frequency of transgenic founders that carry knockins is lower than the number of pups that carry deletions at the site of the double strand break in the chromosome. Efforts to increase the frequency of homology directed repair have centered on the use of the SCR7 small molecule inhibitor of NHEJ (Maruyama et al., 2015, Singh et al., 2014). The Transgenic Core has used SCR7 in experiments, however all oligo knockins and plasmid knockins produced in the Core have not required the use of SCR7 to reduce non-homologous end-joining repair of double strand breaks.

What to expect in your CRSIPR/Cas9 edited founder animal. The frequency of homologous recombination with a plasmid donor is lower than the frequency of indels produced by non-homologous endjoining (Yang et al., 2014). It is our experience and that of others that the founders animals from Cas9 experiments are mosaic. Cas9 is microinjected into fertilized eggs (one cell eggs). Cas9 protein and sgRNA are produced in the egg (one cell) and then persist in the cell as it divides to two, four, eiight, 16, 32, 64 cells three days later. Thus if the Cas9 induces a double strand break in multiple independent cells at the 16 or 32 cell stage embryo the resulting animal will carry more than one allele for the targeted gene. For example, Shen et al. (2013, Fig. S9) subjected genomic DNA from a single founder animals derived by Cas9 gene targeted of EGP to PCR amplification, cloned the PCR product into plasmids, and obtained DNA sequences from 50 cloned DNA fragments. They observed the wild type and six mutant alleles in the genomic DNA of a single founder for a total of 7 different versions of the EGFP gene in a single mouse. As a result is not unusual for a single founder animal to transmit more than one allele to its N1 progeny after mating with a wild type breeding partner. It is also not unusual for all of the N1 pups from such a mating to inherit one mutant allele (from the founder) and one wild type allele.

It is unlikely that all of the N1 littermates will carry the same mutant allele. At this stage it is essential to verify the mutations in the N1 animals by DNA sequence analysis before proceeding with the N2 generation. If the mutation is in-frame (e.g. the deletion of a single codon) gene function may not be knocked out. Ideally the analysis of homozygous animals carrying the same knockout mutation on both chromosomes will be used to assess gene function.

Summary

In summary, the Transgenic Core CRISPR/Cas9 Pipeline includes the following steps:

1. Investigator identifies gene of interest to the Core
2. Investigator identifies upstream exon common to all gene isoforms for a knockout, or a genomic location for a knockin to the Core
3. Transgenic Core uses Broad Institute Design sgRNAs for CRISPRko to find sgRNAs for use with Cas9.
4. Transgenic Core clones two sgRNAs into Cas9 plasmids.
5. Transgenic Core designs a genotyping assay to detect Cas9 activity.
6. Transgenic Core co-electroporates plasmids with a puro-resistance plasmid into mouse ES cells or other appropriate cell line.
7. Transgenic Core purifies genomic DNA from surviving cells and tests for double strand breaks with a Cel I assay.
   
   Activity of Cas9 (Cas9 Image)
8. Core purifies active CRISPR/Cas9 plasmid for microinjection and microinjects it into fertilized rat or mouse eggs at 5 nanograms/microliter. (Mashiko et al., 2014). See RNA Microinjection Buffer protocol.
9. Transgenic Core or Investigator screens for mutant founders by PCR and the Cel I assay or by DNA sequencing
10. Investigator breeds mutant founder mice or rats with wild type mating partners. The mutant founders usually carry multiple mutations of the gene of interest because the Cas9 or Cas9n protein produces chromosome breaks after the microinjected egg has divided several times. Cloning the mutations from the founders that may not be transmitted to offspring is not essential. Cloning the mutations takes place after germline transmission.
11. Investigator clones out the genes from pups produced by the mating of mutant founders and wild type animals to identify the exact DNA sequence of the mutation and to verify transmission of the desired mutation.
12. If desired, Investigator intercrosses heterozygous animals to obtain homozygous mutants for phenotypic characterization.

Obtaining CRISPR/Cas9 Genomic Edited Mice and Rats

The Transgenic Core routinley generates new mouse or rat models with CRISPR/Cas9 for biomedical research and guarantees mutants carrying indels produced by non-homologous end joining. If you need a novel rodent model with genetic modifications to establish a pre-clinical model or to test a biological hypothesis, you need only submit a request at our electronic portal. The Transgenic Core team will provide you with a genetic engineering design for your review. Once approved, we will take all necessary steps to work with you to produce transgenic mouse or rat founders for your laboratory.

Unlike conventional transgenic mice, we find that founders produced with CRISPR/Cas9 technology are often genetic mosaics. That is to say that instead of simply being transgene positive or transgene negative a single G0 founder may include in its tissues cells that carry the wild type gene, the gene with the desired knockin, the gene with an insertion/deletion (indel) caused by nonhomologous end joining of CRISPR/Cas9 induced chromosome breaks, and multiple other indels that are genetically distinct (as many as six). Some indels may cause amino acid deletions; others may introduce premature termination codons. Thus the effect of each indel on protein function must be assessed individually.

Identifying G0 Founder Mice or Rats

To identify G0 founders with the desired genome editing event it is often necessary to use TOPO TA cloning of the G0 founders genotyping PCR products, or to use next-generation DNA sequencing (NGS) approaches. Positive G0 founders are mated with wild type animals to obtain germline transmission of the desired allele. An alternative strategy is to forgo TOPO TA cloning of G0 founders and to simply mate a number of founders that show promising DNA sequence chromatograms with wild type mice. The G1 pups of a mosaic G0 founder will be obligate heterozygotes. That is to say they can only inherit one of the alleles from the mosaic G0 founder and the corresponding wild type allele from their mating partner. It is inadvisable to intercross mosaic G0 founders because of the unpredictability of the genotypes that will be transmitted. DNA chromatograms of the region of interest can be analyzed by subtracting the base pairs of the expected wild type sequence from the chromatogram and thus reveal the DNA sequence of the mutation that was transmitted by the G0 founder. TOPO TA cloning or NGS should be used to validate predicted sequences deduced from chromatograms with overlapping peaks.

Ideally, genotyping PCR assays are established before G0 mosaic founders are born. PCR primers can be tested with wild type DNA or with wild type DNA that has been mixed with an artificial DNA synthesized template to mimic the desired genome editing event. See the copy standards page for more information on setting up artificial gene edited mouse templates for genotyping assay verification. PCR reactions should produce a single band on an agarose gel. The amplicon from the genotyping PCR should be purified and submitted for DNA Sanger sequencing. Please note that the DNA sequence file is often uninformative because the G0 founder mice are often mosaic. Evidence of genome editing will be present in the DNA chromatogram. The presence of multiple alleles in G0 mosaic founders that result from CRISPR/Cas9 NHEJ repair produces overlapping sequences that appear as peaks on peaks in chromatograms. The interpretation of the chromatograms provides the evidence to indicate that the desired genome editing event has occurred.

The Transgenic Core uses the Qiagen QIAquick PCR Purification Kit to prepare amplicons for Sanger Sequencing. If necessary, PCR products can be isolate as bands from an agarose gel for DNA sequencing analysis. Information on submitting samples to the Advanced Genomics Core and software that can be used to view sequencing chromatograms are available on their website.

Primer Design Suggestions for Specific and Sensitive PCR Assays.
Use Primer-Blast to pick primers
http://www.ncbi.nlm.nih.gov/tools/primer-blast
Adjust Primer Parameter default settings
Minimum primer melting temperature: change to 60°C
Optimal primer melting temperature: change to 63°C
Maximum primer melting temperature: change to 66°C
Minimum primer melting temperature difference: change to 1°C
Adjust Specificity Checking Parameters
Click box to turn on “Enable search for primer pairs specific to the intended PCR template”
Set Search Mode to “Automatic”
Set Database to “Genome (reference assembly from selected organisms)”
Set Organism to “Mus musculus (taxid: 10090)”

Click on “Advanced Parameters”
Set Primer Size Min to 27
Set Primer Size Opt to 29
Set Primer Size Max to 31

Stratman et al. reported primers of 27-30 nucleotides made up of 50-60% GC content and that will produce a 100-500 bp PCR product uniformly detect genomic templates with single copy sensitivity.

PCR Enhancer Mix
The 5X CES PCR enhancer mix described by Ralser et al. can improve the results obtained from difficult PCR reactions. The authors further recommend that the PCR reaction buffer contain the following final concentrations of components:
65 mM Tris–HCl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20 at a pH of 8.8.

5X CES
2.7 M betaine
6.7 mM DTT
6.7% DMSO
55 ug/ml BSA

Selected Bibliography

Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE.2014. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol.32:1262-1267.
Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. 2015. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 33: 179-186.

Fujii W, Onuma A, Sugiura K, Naito K. 2014. Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system. Biochem Biophys Res Commun. 445:791-794.

Haeussler M, Schönig K, Eckert H, Eschstruth A, Mianné J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP. 2016. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17:148.

Gabriel R, von Kalle C, Schmidt M. 2015. Mapping the precision of genome editing. Nat Biotechnol. 33: 150-2.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X,Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 31:827-382.

Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. 2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotech. 2015 Mar 23. [Epub ahead of print]

Mashiko D, Young SA, Muto M, Kato H, Nozawa K, Ogawa M, Noda T, Kim YJ, Satouh Y, Fujihara Y, Ikawa M. 2014. Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev Growth Differ. 56:122-129.

Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006 Sep 1;347(3):747-51.

Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. 2013a. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8:2281-2308.

Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. 2013b. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell. 154:1380-1389.

Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. 2014. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 4:5400.

Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X. 2013 Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.. Cell Res. 23:720-723.

Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC. 2014. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods. 11: 399-402.

Singh P, Schimenti JC, Bolcun-Filas E. 2014. A Mouse Geneticist's Practical Guide to CRISPR Applications. Genetics. 199: 1-15.

Stratman JL, Barnes WM, Simon TC. 2003. Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res. 12:521-522.

Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK. 2015. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 33:187-197.

Wang X, Wang Y, Wu X, Wang J, Wang Y, Qiu Z, Chang T, Huang H, Lin RJ, Yee JK. 2015. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol. 33:175-178.

• Space in the ES cell lab can be provided to labs that want to produce targeted ES cells.
• Microinjection workstations are available for labs that want to do their own injections.
• Adherent cell microinjection workstation equipped with Eppendorf Injectman System for Microinjection of Adherent Cells. This consists of a Model 5179 Micromanipulator and a FemtoJet 5247 Microinjector.
• Conversion of Mice to Specific Pathogen Free Status by embryo transfer.
• Mouse strain cryopreservation.
• Recovery of Cryopreserved Mouse Embryos.
• Consulting on transgene design, targeting vector design, and experimental plan.
• Specialized research projects that take advantage of our equipment and expertise such as intraplacental microinjection.

• We guarantee the production of transgenic mice, but can not guarantee that any given transgene will be expressed in transgenic mice.
• We guarantee a minimum number of blastocysts will be injected with ES cells, but can not guarantee that any given ES cell clone will form germline mouse chimeras.
• We guarantee that the gene targeting reagents are germline competent.
• The Genetically engineered animals provided by this facility can only be used for research purposes.
• Access to cell lines and plasmids is only provided to investigators who have complete, approved material transfer agreements.
• Investigators must have approval to use animals in research and teaching from the Institutional Animal Care & Use Committee.

The Transgenic Core has received permission from Dr. David Gordon to distribute the p-loxp-2FRT-PGKneo plasmid. It contains a single loxP site adjacent to a PGKneo cassette that is flanked by FRT sites. This allows you to construct a conditional gene allele in the following way:

1) insert a loxP site on the 5′ or 3′ end of your target exon (avoid splicing sequences and conserved non-coding sequences or other sequence important for normal gene expression)

2) insert this cassette on the opposite side of the target exon ((avoid splicing sequences and conserved non-coding sequences or other sequence important for normal gene expression). 3) make sure that the loxP sites are in the same orientation or excision will not occur. FLP mediated excision will then remove the PGKneo cassette and leave behind a single FRT site. Cre mediated excision will remove the targeted exon and the FRT site.

Before we can distribute p-loxp-2FRT-PGKneo we need you to FAX us a brief letter addressed to Dr. Gordon stating that the p-loxp-2FRT-PGKneo vector will be used for research purposes only. This will allow us to track how many people are using his vector. All publications which include data derived from the use of this plasmid should acknowledge Dr. Gordon for making p-loxp-2FRT-PGKneo available. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number and we will send you the plasmid. An example letter follows:

Download ploxP-2FRT-PGKneo MAP

Download ploxP-2FRT-PGKneo SEQUENCE

Dr. David Gordon
University of Colorado Health Science Center
Endocrinology and Metabolism and Diabetes
Biomedical Research Building
4200 East Ninth Avenue, Box B151
Denver, Colorado 80262

Dear Dr. Gordon,

We wish to use the p-loxp-2FRT-PGKneo targeting vector in our gene targeting research project. The vector will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid.

Sincerely,

Jane or Joe P.I.

The Transgenic Core has received permission from Dr. James Shayman to distribute the ploxPFLPneo plasmid. This plasmid contains two loxP sites and a neo cassette flanked by FRT sites.  Thanks go to Dennis Larkin for the annotated ploxPFLPneo map. Use these plasmids to construct a conditional allele by placing your target exon between the two loxP sites. Subsequently FLP mediated excision will delete the neo cassette, leaving behind a single FRT site. Do not position any of the elements in the plasmid in splicing sequences or conserved non-coding sequences or other sequence important for normal gene expression. Cre mediated expression will then delete the targeted exon and leave behind a loxP site and a FRT site.

Before we can distribute the ploxPFLPneo plasmid we need a brief letter addressed to Dr. Shayman stating that the ploxPFLPneo plasmid will be used for research purposes only. This will allow us to track how many people are using his vector. All publications which include data derived from the use of this plasmid should acknowledge Dr. Shayman for making the plasmid available and reference this paper: Hiraoka M, Abe A, Lu Y, Yang K, Han X, Gross RW, Shayman JA. 2006. Lysosomal phospholipase A2 and phospholipidosis. Mol Cell Biol. 26:6139-48. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number and we will send you the plasmid. An example letter follows:

Download ploxPFLPneo MAP

Download ploxPFLPneo SEQUENCE

Dr. James Shayman
University of Michigan Medical School
Department of Internal Medicine
1150 West Medical Center Drive
Ann Arbor, MI 48109

Dear Dr. Shayman,

We wish to use the ploxPFlpneo targeting vector in our gene targeting research project. The vectors will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid.

Sincerely,

Joe P.I.

The Transgenic Core has received permission from Dr. Francis Stewart to distribute the pOG-Flpe6 plasmid. The plasmid was described in Buchholz F, Angrand PO, Stewart AF. 1998. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 1998 Jul;16(7):657-662. See also http://www.biologie.tu-dresden.de/stewart/index.htm Here is his note regarding this plasmid:

“If you are receiving pOG-Flpe6, please note the three following points –

“1. One of the 4 a.a changes in Flpe as published (P2S) is not in pOG-Flpe6. It was complicated to clone this into the pOG vector so, under pressure to get the revised version back to the journal (the referees wanted a functional test in a mammalian cell line) we tested pOG-Flpe6 without this change and found it to be the best vector (of 6 variations) in both 293 and ES cells. We think that P2S is probably a mutation that was selected in our E. coli-based evolution strategy because it conveys increased protein stability on Flp in E. coli. This is because it is known that the initiating methionine of Flp is processed off in E. coli exposing it to the N-terminal protein stability rules defined by Varshavsky. Also, the N-terminae of other Flp-type recombinases is quite variable and it is unlikely that this region plays any functional role. We are using this plasmid to delete FRT flanked selectable markers in ES cells.”

“2. I ask for one consideration from all who receive Flpe. We are currently generating, hopefully, a Flp deletor mouse line and are trying both pOG-Flpe6 and phubac-Flpe6. If you make a Flp deletor line (i.e. a mouse line that expresses Flpe so that it can be crossed to other lines to remove FRT cassettes in a ubiquitous manner), then I ask that this be collaborative with my lab. Any other use of Flpe is entirely yours. Obviously you can ignore this request, but I think it reasonable – I want to guarantee value to those in my lab doing this Flp deletor work.”

“3. These reagents are completely available to anybody. You are free to pass them on without any need to ask permission. (But for Flpe, please mention point 2).”

Before we can distribute pOG-Flpe6  we need a brief letter addressed to Dr. Stewart stating that the pOG-Flpe6 vector will be used for research purposes only. We will use your letter to track how many people are using his vector. All publications which include data derived from the use of this plasmid should acknowledge Dr. Stewart for making pOG-Flpe6 available. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number and we will send you the plasmid. An example letter follows:

Download pOG-Flpe6 Sequence

Download pOG-Flpe6 MAP

Dr. A. Francis Stewart, Ph.D.
University of Technology, Dresden
Biotec, Genomics
Tatzberg 47 – 51
01307 Dresden
Germany

I wish to use the pOG-Flpe6 targeting vector in my research . The vector will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid. I will reference Buchholz et al., 1998 regarding the vector.

Sincerely,

Joe or Jane P.I.

The Transgenic Core has received permission from Dr. Richard Mulligan to distribute the pPNT gene targeting vector to interested investigators. Details on pPNT construction are in Tybulewicz, VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. 1991. Neonatal Lethality and Lymphopenia in Mice with a Homozygous Disruption of the c-abl Proto-Oncogene. Cell 65:1153-1163.

Before we can distribute pPNT we need a brief letter addressed to Dr. Mulligan stating that the pPNT vector will be used for research purposes only. We will use your letter to track how many people are using his vector. All publications which include data derived from the use of this plasmid should acknowledge Dr. Mulligan for making pPNT available. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number and we will send you the plasmid. An example letter follows:

Download pPNT/pPN2T/pPN2T-HGterm SEQUENCES

Download pPNT MAP

Dr. Richard Mulligan
Children’s Hospital
300 Longwood Avenue
Boston, MA 02115

Dear Dr. Mulligan,

We wish to use the pPNT targeting vector in our gene targeting research project. The vector will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid. I will reference Tybulewicz et al., 1991 regarding the vector.

Sincerely,

Joe or Jane P.I.

The Transgenic Core has received permission from Dr. Chris Paszty to distribute the pPN2T and pPN2T-HGterm vectors to interested investigators. Details on pPN2T vector construction are in Paszty C, Mohandas N, Stevens ME, Loring JF, Liebhaber SA, Brion CM, Rubin EM. 1995. Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue. Nat Genet. 11:33-39 and pPN2T-HGterm construction is described in Zhu Y, Paszty C, Turetsky T, Tsai S, Kuypers FA, Lee G, Cooper P, Gallagher PG, Stevens ME, Rubin E, Mohandas N, Mentzer WC. 1999. Stomatocytosis is absent in “stomatin”-deficient murine red blood cells. Blood. 93:2404-2410. Before we can distribute the pPN2T and pPN2T-HGterm vectors we need a brief letter addressed to Dr. Paszty stating that the pPN2T and pPN2T-HGterm vectors will be used for research purposes only. This will allow us to track how many people are using his vectors. All publications which include data derived from the use of this plasmid should acknowledge Dr. Paszty for making the plasmids available. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number na d we will send you the plasmid. An example letter follows:

Download pPNT/pPN2T/pPN2T-HGterm SEQUENCES

Chris Paszty
Amgen Inc.
Dept. of Molecular Genetics
One Amgen Center, MS 14-1-B
Thousand Oaks, CA  91320-1789

Dear Dr. Paszty,

We wish to use the pPN2T  and pPN2T-HGterm targeting vectors in our gene targeting research project. The vector will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid. I will reference the appropriate publication describing the vector(s).

Sincerely,

Jane or Joe P.I.

The Transgenic Core has received permission from Richard Palmiter to distribute the pnlacf plasmid to interested investigators. The pnlacf plasmid has a nuclear localized beta galactosidase gene that can be used as a reporter gene.

Before we can distribute pnlacf we need a brief letter addressed to Dr. Palmiter stating that the pnlacf plasmid will be used for research purposes only. All publications which include data derived from the use of this plasmid should acknowledge Dr. Palmiter for making pnlacF available.  Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number na d we will send you the plasmid. An example letter follows:

Download pnlacf SEQUENCE

Download pnlacf MAP

Dr. Richard Palmiter
University of Washington
Howard Hughes Medical Institute
Box 357370
Seattle, Washington 98195-7370

Dear Dr. Palmiter,

We wish to use the pnlacf plasmid in our research project. The plasmid will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid.

Sincerely,

Joe or Jane P.I.

The Transgenic Core has received permission from Dr. Steven Domino to distribute the pnlacfNotINeo gene targeting vector to interested investigators. Details on pnlacfNotINeo construction are in Domino SE, Zhang L, Gillespie PJ, Saunders TL, Lowe JB. Deficiency of reproductive tract alpha(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 alpha(1,2)fucosyltransferase locus. Mol. Cell Biol. 21:8336-45.

Before we can distribute pnlacfNotINeo we need a brief letter addressed to Dr. Domino stating that the pnlacfNotINeo vector will be used for research purposes only. We will use your letter to track how many people are using his vector. All publications which include data derived from the use of this plasmid should acknowledge Dr. Domino for making pnlacfNotINeo available. Our FAX number is 734-936-2622. Include your mailing address, telephone number and Federal Express account number and we will send you the plasmid. An example letter follows:

Download pnlacfNotINeo SEQUENCE

Dr. Steven Domino
Department of Obstetrics and Gynecology
6428 Medical Science Bldg. I
1150 West Medical Center Dr.
The University of Michigan
Ann Arbor, MI 48109-0617

 E-mail: [email protected].

Dear Dr. Domino,

We wish to use the pnlacfNotINeo targeting vector in our gene targeting research project. The vector will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid. I will reference Domino et al., 2001 regarding the vector.

Sincerely,

Joe or Jane P.I.

For University of Michigan Investigators: The Transgenic Core has received permission from Richard Palmiter to distribute the pNZTK2 targeting vector to interested investigators. This vector has neor, HSV-TK, and beta- galactosidase cassettes. It is designed to facilitate the construction of gene targeting vectors that place beta-galactosidase under the control of the endogenous gene promoter. Before we can distribute pNZTK2, the investigator needs to contact Dr. Palmiter and obtain a completed materials transfer agreement from him. Simply send a brief letter addressed to Dr. Palmiter stating that the pNZTK2 plasmid will be used for research purposes only. All publications which include data derived from the use of this plasmid should acknowledge Dr. Palmiter for making pNZTK2 available. An example letter follows:

For Other Investigators: Contact Dr. Palmiter Directly and ask him to send you the plasmid.

Download pNZTK2 Partial Sequence

Download pNZTK2 Map

Dr. Richard Palmiter                          FAX (206) 543-0858
University of Washington
Howard Hughes Medical Institute
Box 357370
Seattle, Washington 98195-7370

Dear Dr. Palmiter,

We wish to use the pNZTK2 plasmid in our research project. The plasmid will be used for research purposes only. I agree to acknowledge and thank you as the source of the plasmid.

Sincerely,

Jane or Joe P.I.

For University of Michigan Investigators: The Transgenic Core has received permission from Jamey Marth to distribute the pflox vector to interested investigators. It is designed to simplify the insertion of loxP sites in genes. Before we can distribute pflox, investigators need to contact Dr. Marth and obtain a completed materials transfer agreement from him. Simply send a letter addressed to Dr. Marth stating that the pflox vector will be used for research purposes only. I suggest that you FAX him the form letter below after transferring it to your letterhead. Bring your copy of the Material Transfer form to the Transgenic Core and we will provide you with the vector.

For Other Investigators: Contact Dr. Marth Directly and ask him to send you the plasmid.

Download pflox Sequence

Download pflox MAP

Dr. Jamey Marth                                                    FAX (858) 534-6724
Professor of Cellular and Molecular Medicine
Associate Investigator, Howard Hughes Medical Institute
University of California San Diego
Howard Hughes Medical Institute – 0625
9500 Gilman Drive
La Jolla, California 92093-0625

Dear Dr. Marth,

I wish to use the pflox vector in my research. It will not be used for commercial purposes and I will not distribute it without your consent. I will acknowledge you as the source of the pflox vector in any publications resulting from its use. I will cite your paper “Chui D, Oh-Eda M, Liao YF, Panneerselvam K, Lal A, Marek KW, Freeze HH, Moremen KW, Fukuda MN, Marth JD. 1997. Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 90:157-167.” Please send me an HHMI Materials Transfer Agreement so that I may complete it and receive the plasmid from Dr. Thom Saunders at the University of Michigan Transgenic Animal Model Core.

Sincerely yours,

Joe or Jane P.I.

For University of Michigan Investigators: The Transgenic Core has received the pMC-Cre vector from Dr. Klaus Rajewsky to distribute to interested investigators. It is designed for the expression of the Cre enzyme in cells and carries a nuclear localization signal. Before we can distribute pMC-Cre, investigators need to contact Dr. Dr. Klaus Rajewsky and obtain permission from him to use pMC-Cre. Simply send a letter addressed to Dr. Rajewsky stating that the pMC-Cre vector will be used for research purposes only and that the University of Michigan has obtained permission to use Cre/LoxP materials for research purposes. I suggest that you FAX him the form letter below after transferring it to your letterhead. Bring your copy of the Material Transfer form to the Transgenic Core and we will provide you with the vector.

For Other Investigators: Contact Dr. Rajewsky Directly and ask him to send you the plasmid.

Download pMC-Cre Sequence

Download pMC-Cre MAP

Dr. Klaus Rajewsky
Professor of Pathology
Harvard Medical School
The CBR Insitute for Biomedical Research
Warren Alpet Building
200 Longwood Ave.
Boston, MA 02115

FAX: 617-278-3129

Dear Dr. Rajewsky,

I wish to use the pMC-Cre vector in my research. It will not be used for commercial purposes and I will not distribute it without your consent. I will acknowledge you as the source of the pMC-Cre vector in any publications resulting from its use. I will cite the paper “Gu H, Zou YR, Rajewsky K. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell. 73:1155-1164.” The University of Michigan has obtained permission from the Du Pont company to use Cre/LoxP technology for research purposes. Please send me an materials transfer agreement so that I may complete it and receive the plasmid from Dr. Thom Saunders at the University of Michigan Transgenic Animal Model Core.

Sincerely yours,

Joe or Jane P.I.

Useful information on the application of Cre recombinase technology can be found at Andras Nagy’s website.

For University of Michigan Investigators: The Transgenic Core has received the pACN vector from Dr. Mario Capecchi. It is designed to express the Cre recombinase and to then delete itself by Cre mediated recombination in the cells  which express it. Please contact Dr. Mario Capecchi to obtain permission from him to use pACN in  your research. Simply send a letter addressed to Dr. Capecchi stating that the pACN vector will be used for research purposes only. I suggest that you FAX him the form letter below after transferring it to your letterhead. Bring your copy of the Material Transfer form to the Transgenic Core and we will provide you with the vector.

For Other Investigators: Contact Dr. Capecchi Directly and ask him to send you the plasmid.

Download the pACN MAP

pACN Sequence

Dr. Mario Capecchi                          Phone: 801-581-4422
Department of Human Genetics
Eccles Institute of Human Genetics
University of Utah
15 North 2030 East, Room 2100
Salt Lake City, UT 84112-5330

Dear Dr. Capecchi,

I wish to use the pACN vector in my research. It will not be used for commercial purposes and I will not distribute it without your consent. I will acknowledge you as the source of the pflox vector in any publications resulting from its use. I will cite your paper Bunting M, Bernstein KE, Greer JM, Capecchi MR, Thomas KR. Targeting genes for self-excision in the germ line. Genes Dev. 1999 Jun 15;13(12):1524-8. Please send me an HHMI Materials Transfer Agreement so that I may complete it and receive the plasmid from Dr. Thom Saunders at the University of Michigan Transgenic Animal Model Core.

Sincerely yours,

Joe or Jane P.I.

The Transgenic Core generated Pat5 ES cells from 129X1/SvJ mice. To date three different genes have been targeted in Pat5 ES cells and published (Chen et al., 2002; Domino et al. 2001). This mouse embryonic stem cell line is available for use at low passage. The Pat5 cell line was isolated from a blastocyst obtained by  mating 129X1/SvJ mice from The Jackson Laboratory. Immunosurgery was performed on the blastocyst to eliminate trophectoderm cells as described (Solter and Knowles, 1975). The inner cell mass cells were established in tissue culture on feeder cells prepared from primary mouse embryo fibroblasts. Pat5 ES cells are cultured in high glucose Dulbeccoís Minimal Essential Medium, supplemented with 15% fetal bovine serum, 1 uM beta-mercaptoethanol, 4 mM glutamine, penicillin (50 I.U. /ml), streptomycin (50 ug /ml) and 1000 U/ml recombinant leukemia inhibitory factor (ESGRO, Chemicon). Electroporation of Pat5 ES cells with the targeting vector and selection for clones was carried essentially as described in Kendall et al. (1995).

Chen C, Bharucha V, Chen Y, Westenbroek RE, Brown A, Malhotra JD, Jones D, Avery C, Gillespie PJ 3rd, Kazen-Gillespie KA, Kazarinova-Noyes K, Shrager P, Saunders TL, Macdonald RL, Ransom BR, Scheuer T, Catterall WA, Isom LL. Reduced sodium channel density, altered voltage dependence of inactivation, and increased susceptibility to seizures in mice lacking sodium channel beta 2-subunits. Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):17072-7.

Domino SE, Zhang L, Gillespie PJ, Saunders TL, Lowe JB. Deficiency of reproductive tract alpha(1,2)fucosylated glycans and normal fertility in mice with targeted deletions of the FUT1 or FUT2 alpha(1,2)fucosyltransferase locus. Mol Cell Biol. 2001 Dec;21(24):8336-45.

Kendall, SK, Samuelson, LC, Saunders, TL, Wood, RI, Camper, SA. 1995. Targeted Disruption of the Pituitary Glycoprotein Hormone alpha-subunit Produces Hypogonadal and Hypothyroid Mice. Genes Dev. 9:2007-2019.

Solter D, Knowles BB. 1975. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 72:5099-102.

If you would like to obtain Pat5 ES cells from please read the Pat5 Material Transfer Agreement and contact [email protected] about finalizing the transfer.

The mouse embryonic stem (ES) cell line R1 was developed in the laboratories of Drs. Janet Rossant and John Roder at Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Canada. This cell line was derived from a cross between 129X1/SvJ and 129S1/Sv-+p+Tyr-cKitlSl-J/+ mice. The derivation of the R1 cell line is described in: Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. 1993. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. 90:8424-8428.

For University of Michigan Investigators: We have streamlined the Material Transfer Agreement process for University of Michigan investigators for the R1 mouse embryonic stem cells. It is still necessary for each investigator to complete a University Material Transfer Form (MTF). The MTF should be completed and signed by the University investigator then sent through the signature approval pathway along with Schedule A to the original Material Transfer Agreement executed between Mt. Sinai and UM. Schedule A should also be signed by the investigator. Upon its arrival at the Division of Research Development and Administration (DRDA) along with the MTF, Schedule A will be signed on behalf of the University and sent to Terry Donaghue at Mt. Sinai. All publications which include data derived from the use of this cell line should acknowledge the contribution of Dr. Andras Nagy, Reka Nagy and Dr. Wanda Abramow-Newerly for making the R1 cell line available.

For Other Investigators: Investigators from other institutions who wish to utilize the R1 ES cells will need an agreement between Mt. Sinai and their respective institutions. Investigators interested in using the R1 cells should write to Mr. Terrence P. Donaghue to arrange for permission and the signing of a materials transfer agreement.

The letter should be addressed to:

OFFICE OF TECHNOLOGY TRANSFER & INDUSTRIAL LIAISON
MOUNT SINAI HOSPITAL
SAMUEL LUNENFELD RESEARCH INSTITUTE
Michael Hanna, Licensing Associate
600 University Ave.
Toronto, Ontario, Canada M5G 1X5
Tel. 416.586.4800 ext. 4752
Fax 416.586.8844
E-mail: hanna(at)mshri.on.ca

The Transgenic Core has received permission from Dr. Thomas Gridley to distribute the CJ7 embryonic stem cell line to interested investigators for research use only. This cell line was derived from 129S1/SvImJ mice. All publications which include data derived from the use of this cell line should acknowledge Dr. Gridley for making CJ7 available. Before we can distribute CJ7 cells we need a brief letter addressed to Dr. Gridley stating that they will be used for research purposes only. This will allow us to track demand for the CJ7 ES cells An example letter follows:

Dr. Thomas Gridley
The Jackson Laboratory
600 Main Street
Bar Harbor, Maine 04609

Dear Dr. Gridley,

We wish to use the CJ7 embryonic stem cell line in our research project. The animals that we generate will be used for research purposes only. I agree to acknowledge you as the source of the cells and reference Swiatek, PJ, and Gridley T. 1993. Perinatal Lethality and defects in hindbrain development in mice homozygous for a targeted mutation of the zinc finger gene Krox20. Genes and Development 7:2071-2084 as the source of the cells.

Sincerely,

Joe or Jane P.I.

The Transgenic Core has received E14Tg2a clone4 ES cells from Dr. Bill Skarnes. This cell line was derived from 129P2/OlaHsd mice. We have permission to distribute the E14Tg2a clone4 embryonic stem cell line to interested investigators for research use only. The cells were generously provided to the research community by Dr. Austin Smith at the University of Edinburgh. All publications which include data derived from the use of this cell line should acknowledge Dr. Smith for making E14Tg2a clone4 ES cells  available. Before we can distribute the cells we need a brief letter addressed to Dr. Smith stating that they will be used for research purposes only. This will allow us to track demand for the E14Tg2a clone4 ES cells. An example letter follows:

Dr. Austin Smith
Center for Genome Research
The University of Edinburgh
Roger Land Building
Edinburgh EH9 3JQ
United Kingdom

Dear Dr. Smith,

I wish to use the E14Tg2a clone4 embryonic stem cell line in my research project. The cells will be used for research purposes only. I agree to acknowledge you as the source of the cells.

Sincerely,

Joe or Jane P.I.

For University of Michigan Investigators: The Bruce4 mouse embryonic stem cell line was provided to the Transgenic Core by Colin Stewart. Unlike the other ES cell lines on this page, the Bruce4 cell line was derived from C57BL/6 mice. We have successfully obtained germline transmission for the unmodified Bruce4 cells and also with gene targeted Bruce4 clones. Please contact Dr. Stewart and obtain a material transfer agreement that allows you to use this ES cell line. An example letter follows.

For Other Investigators: Obtain the Bruce4 ES cell line directly from Dr. Stewart.

Dr. Colin Stewart
Cancer and Developmental Biology Laboratory
NCI-FCRDC
Building 539, Room 121A
P.O. Box B
Frederick, MD 21702-1201

FAX 301- 846-7117

Dear Dr. Stewart,

We wish to use the Bruce4 embryonic stem cell line in our research. It  will be used for research purposes only. I agree to acknowledge you as the source of the cells and to reference Kontgen F, Suss G, Stewart C, Steinmetz M, Bluethmann H. 1993. Targeted disruption of the MHC class II Aa gene in C57BL/6 mice. Int Immunol. Aug;5(8):957-964 as the origin of the cell line.

Sincerely,

Jane or Joe P.I.

The Transgenic Core has received permission from Dr. Thomas Doetschman to distribute the D3 embryonic stem cell line to interested investigators for research use only. This cell line was derived from 129S2/SvPas mice. All publications which include data derived from the use of this cell line should acknowledge Dr. Doetschman for making D3 available. Before we can distribute D3 cells we need a brief letter addressed to Dr. Doetschman stating that they will be used for research purposes only. This will allow us to track demand for the D3 ES cells. An example letter follows:

Dr. Thomas Doetschman
Department of Molecular Genetics
College of Medicine
University of Cincinnati
Cincinnati, OH 45267

Dear Dr. Doetschman,

We wish to use the D3 embryonic stem cell line in our research. It and any gene targeted animals produced will be used for research purposes only. I agree to acknowledge you as the source of the cells and to reference Doetschman, TC, Eistetter, H, Katz, M, Schmidt, W, Kemler, R. 1985. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87:27-45 as the origin of the cell line.

Sincerely,

Jane or Joe P.I.

Transgenic and gene “knockout” animals that have been developed using NIH IRP (intramural research program) funds and resources will be provided to other laboratories following publication of descriptions of the animals in the peer reviewed literature. It is an obligation of NIH intramural scientists to make such animals widely available for research purposes. This can be achieved by making arrangements to send breeding pairs to a central repository such as the Induced Mutant Resource at the Jackson Laboratory. This would assure the availability of clean, genetically characterized animals within a year’s time. An attempt should be made to reduce duplication of effort by setting up collaborative experiments whenever possible; however, this should not be used as a mechanism to inhibit the distribution of animals.

These guidelines for the IRP are now in agreement with those of the US Public Health Service (PHS) for the extramural community: “It is the policy of PHS to make available to the public the results and accomplishments of the activities that it funds…. Therefore, when these resources are developed with PHS funds and the associated research findings have been published or after they have been provided to the agencies under contract, it is important that they be made readily available for research purposes to qualified individuals within the scientific community. This policy applies to grants, cooperative agreements, and contracts.”

These guidelines supplement those already covered by the NIH Guide for other types of biological materials and resources. The NIH Guide for the Care and Use of Laboratory Animals is available online.