connectionconnectionW

inter 2006

ATCC and Roche Applied Science have combined resources to benefit life sci-

ence investigators by consolidating infor-mation on transfection and routinely used cell lines. The partnership will provide effi-ciencies in experimental design and can improve the rate of successful transfection for the life science research community.

The Roche/ATCC partnership links website resources from the two organizations to provide added convenience and enhanced productivity to research laboratories. On

its website (www.roche-applied-sci-ence.com/transfection), Roche offers a searchable database of cell lines trans-fected and ranked using FuGENE® 6 and FuGENE® HD, along with protocols and links to associated peer-reviewed publications.

The database includes protocols and information on the hundreds of ATCC cell lines that have been transfected using FuGENE® 6 and FuGENE® HD transfection reagents, either by Roche

In This Issuep.1Roche and ATCC Transfection

PartnershipCLSI Supports ATCC Standards

Resource™

p. 2What’s New: Cell Biology 2007

Catalog AvailableNew Genomic DNA Products

p.3hTERT Cell Immortalization

p.4Effects of Passage Number on

Transfection

p.5ATCC Seminar at ASCB Meeting

p. 6ELF® Phosphatase Detection

Kit

p. 7Tech Qs: hTERT Immortaliza-

tion; Transfection Protocol

p.8Upcoming Meetings

continues on page 7

Volume 26 Numb er 2

ATCC Standards Resource™ Boosted By Support From CLSI

continues on page 6

Roche Applied Science and ATCC Partner on Cell Line Transfection

A collaboration between ATCC and the Clinical and Laboratory Standards

Institute® (CLSI, formerly NCCLS) greatly enhances the usefulness and value of ATCC’s Web-based Standards Resource for both CLSI members and other laboratory scien-tists in need of standard test methods.

This collaboration enhances the ATCC Stan-dards Resource, providing mutual customers of the two organizations with rapid access to cross-referenced information about widely used microbiology test methods and labo-

ratory procedural standards from CLSI that cite ATCC products.

Part of the wider ATCC standards pro-gram, the Web-based resource is a searchable database of standard test methods and commercial assays that is linked to information on ATCC’s col-lection of reference cultures. The search tool allows scientists and laboratory professionals to determine which ATCC bacteria, viruses, fungi or cell lines are specified in CLSI standards.

2 ATCC Connection

What’s new

More Nucleic Acids

Let ATCC do the isolation for you

If you are still extracting DNA for microbial research, it’s time to let ATCC do the work. Genomic DNA from ATCC’s well-char-acterized microbial strains can save you the time and expense of isolating DNA yourself. ATCC offers high-quality DNA from hundreds of genuine ATCC bacterial, fungal and protozoan strains. Check out ATCC’s new genomic DNA from well-character-ized microbial strains listed below. All are available in the 5-µg package size. ATCC testing has expanded to include the deter-mination of the total amount of DNA by PicoGreen® measurement. This high-quality DNA has been isolated under aseptic conditions to prevent cross-contamination. Batches are evaluated for integrity, purity and quality by several methods, includ-ing agarose gel electrophoresis, spectrophotometry, suitability for amplification by PCR, sequencing of the 16S ribosomal RNA gene (first ~500 base pairs) consistent with the sequence of the source organism for bacteria and ITS sequencing for fungi and yeast. Look for regular additions to this list online at: http://www.atcc.org/common/products/PurifiedDNA.cfm#NA.

ATCC Number Source Organism Source Strain Significance

10895D-5 Ashbya gossypii NRRL Y-1056 BAA-1234D-5 Campylobacter jejuni RM3193 10231D-5 Candida albicans NIH 3147 2001D-5 Candida glabrata CBS 138 Type strain; Sequenced genomeMYA-565D-5 Cryptococcus neoformans JEC 21 Sequenced genome36239D-5 Debaryomyces hansenii CBS 767 Type strain; Sequenced genome700721D-5 Klebsiella pneumoniae MGH78578 Sequencing in subsp. pneumoniae progress 8585D-5 Kluyveromyces lactis MRRL-Y-1140 Sequenced genome23037D-5 Mycobacterium smegmatis 1102 53417D-5 Neisseria meningitidis M1027 Serogroup A11696D-5 Ralstonia solanacearum 60-1 Type strain204508D-5 Saccharomyces cerevisiae S288C Sequenced genomeBAA-1250D-5 Salmonella enterica serovar SPB7 Paratyphi B 29903D-5 Shigella flexneri 24570 Type strain27419D-5 Streptomyces avidinii ISP 5526 Type strain700294D-5 Streptococcus pyogenes SF370, M1 GAS Sequenced genome

Order online or call (800) 638-6597 or (703) 365-2700.Scanning electron microscope image of Campylobacter jejuni .

ATCC released the 2007 Cell Biology Catalog, which highlights the 3,600 cell lines and hybrid-omas that comprise the ATCC cell biology collection, as well as the high-performance media, sera and reagents used to culture ATCC cell lines. New and updated features include:

• An expanded guide to navigating the ATCC website to assist you in searching for cell lines of interest

• A detailed technical section to help boost your cell culture success• Indexes organizing the cell biology collection by species of origin, tissue source, disease

type and other traits, to provide user-friendly searching

By using the 2007 Cell Biology Catalog in conjunction with the ATCC website, you can access comprehensive information about ATCC’s authenticated, low-passage cell lines, which are fully tested for contamination, misidentification and genetic instability.

The ATCC 2007 Cell Biology Catalog is Now Available

3 ATCC Connection

Move over primary cells — hTERT is here!

Primary cells, once the only choice for many research projects, are now

giving way to hTERT-immortalized cell lines. hTERT (human TElomerase Reverse Transcriptase)-immortalized cells offer many advantages over primary cells.

• Enable the use of the same consistent material throughout a research project

• Provide an extended life span while maintaining the genotype and pheno-type markers of the source tissue

• Eliminate lot-to-lot variability common with primary cells

• Reduce experimental costs

Cells that exogenously express hTERT are able to avoid replicative senescence by maintaining sufficient telomere length without altering critical cellular processes such as apoptosis1-6. ATCC currently dis-tributes several immortalized cell lines which have been comprehensively char-acterized and authenticated. ATCC tests the immortalized cell lines to ensure the following:

• Extended proliferative capacity• Stable genotype• Selected phenotypic markers (relative

to cell type) present• Continued expression of hTERT protein

hTERT — a superior cell immortaliza-tion technology

Imagine converting your primary cells into a continuous cell line that maintains important characteristics of the source material and gives you a consistent supply of material for an entire research project. Immortalization of primary cells is a difficult process made easy with the well-tested eukaryotic expression plas-mid (ATCC® catalog no. MBA-141).

To ensure quality, the hTERT plasmid is tested using amaxa Nucleofector® tech-nology to verify transfection capabilities. Following transfection, telomerase activ-ity is confirmed by telomere repeat ampli-fication protocols (TRAP) assay.

Cell immortalization methods that employ viral antigens or oncogenes result in genomic abnormalities and changes in cellular function. Immortalization with hTERT synthesizes and maintains the

telomeres at the end of the chromosomes and helps the cells escape replicative senescence. To a large extent, cells immor-talized with hTERT technology retain the characteristics of the parental tissue and evade some of the difficulties associ-ated with non-hTERT approaches.

References:

1. Hahn WC. Immortalization and transformation of human cells. Mol. Cells. 13(3): 351-361. 2002.

2. Harley CB. Telomerase is not an oncogene. Oncogene. 21(4): 494-502. 2002.

3. Rubin H. The disparity between human cell senescence in vitro and lifelong replication in vivo. Nature Biotechnology. 20(7): 675-681. 2002.

4. Lee KM. Immortalization with telomerase of the Nestin-positive cells of the human pancreas. BBRC. 301: 1038-1044. 2003.

5. Jiang XR. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nature Genetics. 21: 111–114. 1999.

6. Morales CP. Absence of cancer associated changes in human fibroblasts immortalized with telomerase. Nature Genetics. 21: 115–118. 1999.

† These materials are subject to claims under U.S. Patent Nos. 6,261,836 and 6,337,200, other pending patent applications, and foreign counterparts thereof. They are provided under the ATCC® Material Transfer Agreement as posted on the ATCC Website (www.atcc.org) and the terms of the Addendum for Commercial and For-Profit Organizations or the Addendum for Noncommercial and Academic Organizations, which are linked to the product’s ATCC catalog number on the ATCC Website.

The TERT-containing plasmid is not available to com-mercial and for-profit organizations or for work to be conducted under funding from a commercial organiza-tion. However, alternative solutions are possible. Con-tact ATCC for more information.

amaxa and Nucleofector are trademarks of amaxa GmbH.

Firgure 1: Immortalized RPE-1, human endometrium retinal pigmented epithelium cells (ATCC® CRL-4000™), are tested for the epithelial marker, cytokeratin.

ATCC — the leader in immortalized cell lines and products

ATCC immortalized cell lines*†human bone cartilage fibroblast ATCC® CRL-2846™ CHON-001human bone cartilage fibroblast ATCC® CRL-2847™ CHON-002human retinal pigmented epithelium ATCC® CRL-4000™ hTERT RPE-1human foreskin fibroblast ATCC® CRL-4001™ BJ-5tahuman endometrium fibroblast-like ATCC® CRL-4003™ T HESCshuman mammary epithelium ATCC® CRL-4010™ hTERT HME1 (ME16C)

ATCC hTERT vectorplasmid in E. coli GC10, pGRN145 ATCC® MBA-141

* ATCC is continually developing new cell lines and other cell immortalization tools. For more information please visit http://www.atcc.org/common/products/CellImmort.cfm

Product Focus

4 ATCC Connection

An important factor in the success of any transfection experiment is the quality of the cells. As a key experimental compo-nent, the cell line can also be the great-est variable, affecting the reliability and reproduciblity of results.

In transfection experiments, the passage number of the cell lines can affect not only transfection efficiency, but protein expression as well.

To eliminate these concerns, Roche Applied Science recommends using freshly obtained, low-passage cell lines from ATCC.

Cell lines are increasingly being recog-nized as standard experimental reagents and as essential components to ensure reproducible and reliable results in life sci-ence research1-4. Manufacturers of quality life science products routinely use ATCC cell lines for product development and optimization.

The performance of an optimized prod-uct can suffer when used with cell lines of inferior quality.

The data shown below demonstrate low- and high-passage RAW 264.7 (ATCC® TIB-71™) cells transfect equally well, but pro-tein expression is significantly reduced in the high-passage samples. RAW 264.7 (ATCC® TIB-71™) cells were transfected with a plasmid for luciferase expression at passage number 5 (low passage) and 74 (high passge) using FuGENE® HD trans-fection reagent for comparative studies. Three volumes (4, 6 and 10 µL) of the same complex (5:2 ratio of reagent to DNA) were added to all cells. Similar expression levels (top graph) were observed 24 hours post-transfection at either passage number.

However, luciferase expression dropped off significantly 48 hours post-transfec-tion in the high-passage cells. Minimal inhibition of cell proliferation (bottom graph) was observed in low-passage cells with all three volumes of complex. In con-trast, growth inhibition was observed in the high-passage cells when 6–10 µL of the complex was added. This effect on proliferation was not observed when less complex was added. (Data supplied by Roche Applied Science)

References

1 Hartung T et al. Good cell culture practice: ECVAM good cell culture practice task force report 1. ATLA 30: 407-414. 2002.

2 Hay RJ, Cleland MM, Durkin S and Reid YA. Cell line preservation and authentication. In “Animal Cell Culture.” JRW Masters (ed.). J Wiley Inc., Oxford University Press, New York City. 2000.

3 Nardone RM. Eradication of cross-contaminated cell lines: A call to action. Available at: http://www.Biotrac.com/pages/authentication.html. 2006.

4 O’Brien SJ. Cell culture forensics. Proceedings of the National Academy of Sciences USA. 98: 7656-7658. 2001.

Effects of Passage Number on Cell Line Transfection

Product Focus

5 ATCC Connection

Seminar attendees can enter a drawing for an Apple iPod® digital music player and enjoy some refreshments at the same time!

ExhibitionWhen you visit the ASCB exhibit hall, don’t forget to stop by the ATCC booth (no. 1301). Fill out a brief survey at the ATCC booth and enter the iPod® drawing!

iPod® is a trademark of Apple Computer, Inc.

ATCC has participated in annual meetings of the American Society for Cell Biology for the past 25 years. Please come and see us again this year. We look forward to seeing old friends and making new ones.

SeminarSee revealing data from peer-reviewed papers on Sunday, December 10 at 6:15 p.m. during a 45-minute seminar. This is a seminar that is sure to change the way you think about cell lines. You can’t afford to miss this – check your meeting brochure for the seminar location.

ATTN: Scientists attending ASCB in San Diego 2006

Don’t miss the ATCC seminar and visit the

ATCC booth (#1301).

6 ATCC Connection

The ATCC ELF® Phosphatase Detection Kit for Embryonic Stem Cells offers a reliable, sensitive and stable assay to determine the state of embryonic stem cell (ESC) differentiation. Using fluorescent detection of endogenous phosphatase activity in embryonic stem cells, researchers can confidently proceed with experiments knowing that their stem cell populations have been maintained in the undifferentiated state. Embryonic stem cell lines are more sensitive to culture conditions than other cell lines and exhibit a tendency to differentiate. ATCC stem cell scientists recommend running the ELF phosphatase assay before starting an experiment when undifferentiated stem cells are required. It is also recommended to run this assay any time cultures have been significantly manipulated or if an unexpected change in morphology occurs. Methods for assessing differentiation states of ESCs that rely on colorimetric or fast red-violet dye result in higher background levels, poor signal-to-noise ratios and days of additional preparation compared to the ATCC ELF Phosphatase Detection Kit*. The ATCC phosphatase assay rapidly delivers a strong, high-resolution signal and low background, all within two days or less.

The ATCC Phosphatase Detection Kit for ESCs provides the following advantages:

• Highest resolution detection • Precise localization of fluorescence • More photostability (up to 3 times) • Less susceptibility to signal bleaching • More definitive test results (reduced

over-staining) *Cox WG et al. A high-resolution, fluorescence-based method for localization of endogenous alkaline phosphatase activity. J. Histochem. Cytochem.; 47(11):1443-56. 1999.

The ELF® Phosphatase Detection Kit for Embryonic Stem Cells: The fastest, most effective way to a successful experiment

Rochecontinued from p. 1

—

Human ES cells, BG01V (ATCC® SCRC-2002™) surrounded by feeder cells are fully undifferentiated as evidenced by the presence of continuous green flourescence throughout the cell mass.

scientists or through peer-reviewed publi-cations. The ATCC website (www.atcc.org) provides detailed information on those cell lines and contains links to the trans-fection information on Roche’s website. Several hundred FuGENE®-transfected ATCC cell lines are available for purchase through the ATCC website. ATCC’s online search engine makes it easy to locate indi-vidual cell lines by their common names or by ATCC catalog number.

“Combining the strengths of our organ-izations through this agreement cre-ates a powerful offering for life sciences research,” said Lonnie Shoff, Senior Vice President, Roche Applied Science. “Sci-entists across the research spectrum — academia, biotech companies, disease research, pharmaceuticals, government, and private institutions — will have the ability to link a transfection protocol, peer-reviewed publications and results information to an ATCC cell line of inter-est.”

“ATCC cell lines are recognized as stan-dards among the research community,” commented Michael Gove, Vice President of Marketing and Sales at ATCC. “By com-bining quality transfection reagents like the FuGENE® family, we have increased the chance for successful lab results and helped scientists reach their research goals faster.”

Choose transfection reagents from Roche Applied Science combined with fresh, authenticated cell lines from ATCC to move closer to discovery.

FuGENE® is a registered trademark of Fugent, L.L.C.,

USA

7 ATCC Connection

Tech Qs

TechQs are intended for informational purposes only. ATCC is not responsible for results obtained from the use of this information.

The ELF® Phosphatase Detection Kit for Embryonic Stem Cells: The fastest, most effective way to a successful experiment

“CLSI’s participation with the ATCC Stan-dards Resource makes up-to-date infor-mation on a wide range of CLSI docu-ments easily available,” said Joseph Per-rone, ScD, Vice President for Standards and Certification at ATCC.

“Websites for the two organizations will be conveniently linked so that users can quickly find and purchase procedural standards and the ATCC biological refer-ence materials needed to carry out those standards,” Perrone added.

ATCC cultures are commonly used as stan-dard controls in areas as diverse as clinical laboratory testing, environmental moni-toring and pharmaceutical development. They are also used widely in biological testing for industrial-scale manufacturing of foods, beverages, textiles, paint, cos-metics and personal care products.

Users of the ATCC Standards Resource can enter a specific CLSI standard and the Web tool will return information about the ATCC reference materials necessary for carrying out that procedure. The search tool can be accessed through ATCC at www.atcc.org.

“With ATCC and CLSI working together, there will be mutual benefits for clinical laboratory scientists in a host of fields in which microorganisms are used for test-ing,” said CLSI Executive Vice President, Glen Fine.

Clinical and Laboratory Standards Insti-tute® (CLSI, formerly NCCLS) is a global nonprofit organization whose vision is to be the leader in clinical and laboratory standards to improve the quality of medi-cal care. For more information on CLSI and its global efforts in standardization, visit www.clsi.org.

CLSIcontinued from p. 1

Web Tips

Can I use hTERT to immortalize my primary rat alveolar type II cells?

ATCC has not attempted to immortalize murine cell lines using the hTERT plasmid. Mouse and rat cells typically undergo spontaneous immortalization in vitro, and differ from human cells in that telomerase is more often expressed in fully differentiated somatic cells. We do not know how human telomerase will affect transfection outcomes in murine cell lines. However, an interesting article in Cancer Research suggests that hTERT inhibits “endogenous murine telomerase activity,” leading to telomere shortening, cell crisis and eventual death via apoptosis (Boklan et al. 2002. 62:2104-2114).

Can you recommend a transfection protocol for ATCC® MBA-141?

It is important to keep in mind that transfection protocols vary depending upon several factors, including the host cell line of choice, the type of DNA/vector being introduced, and the transfection reagent(s) or machinery being used during the procedure. While we are currently working with amaxa to provide optimized transfection protocols for some of our cell lines, we have only used MBA-141 to transfect IMR-90 cells (ATCC® CCL-186™). It is important to note, however, that transfections of IMR-90 cells using MBA-141 at ATCC are only transient (using both 2.0 µg and 1.5 µg with success); we have not attempted to perform stable transfections using this vector. If you are interested in using amaxa’s Nucleofector® technology with IMR-90 cells, we used program no. V20 with Nucleofector® Solution V. Additional information pertaining to amaxa’s Nucleofector® technology can be found online at www.amaxa.com.

ATCC Standards Resource Search Tips

How can I use the ATCC Standards Resource to find ATCC cultures specified in a standard test method or commercial protocol?

The Web-based standards database was developed for scientists and laboratory professionals to determine which ATCC bacteria, fungi, viruses or cell lines are specified for use in a particular standardized test method or procedure. You can get to this tool by visiting the ATCC website (www.atcc.org) and clicking on the “ATCC Standards” button. There, you have several search options:

• Search for the name of the organization or company that developed the standard (using a browseable listing).

• Search for the name and/or number of the test or identification system (using a browseable listing).

• Search the full text of a catalog description using keywords. You can narrow your search by selecting one or more fields.

Focus

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8 ATCC Connection

P.O. Box 1549Manassas, VA 20108Tel: 703.365.2700 Toll Free Tel: 800.638.6597www.atcc.org

NOTE: BEI Resources will have a booth at the American Society of Microbiology Biode-fense Research Meeting (February 27-March 2, Washington, DC) and the ASM General Meeting (May 21-25, Toronto, Canada).

This newsletter is published by ATCC and is distributed free of charge upon request. Direct all correspondence to P.O. Box 1549, Manassas, VA, 20108, or e-mail news@atcc.org. Photocopies may be made for personal or internal use without charge. This consent does not extend to copying for general distribution, promotion, creating new works, or resale.

© 2006 ATCC. ISSN 1088-2103 The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection.ELF and Molecular Probes are trademarks of Molecular Probes, Inc. Product is optimized by ATCC and made by Molecular Probes.

These products are for laboratory research use only. They are not intended for use in humans, animals or for diagnostics. ATCC® products may not be resold, modified for resale, used to provide commercial services or to manufacture commercial products without prior ATCC written agreement.

American Association for Cancer Research April 14-18, Los Angeles, CA

Food Safety World Conference & Expo April 19-20, Washington, DC

American Society for Microbiology Meeting May 21-25, Toronto, Canada

Meetings and ConferencesATCC will be attending the following meetings. Stop by and talk to an ATCC representative about how we can help your research.

American Society for Cell Biology Annual MeetingDecember 9-13, San Diego, CA

RMUG - Rapid Micro Users Group January 21-23, Arlington, VA

Mid-Atlantic Microbial Pathogenesis Meeting February 11-13, Wintergreen, VA