• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Authentication of Cell Lines

        互联网

        1565

        Authentication Techniques

        Whatever the scope of work to be carried out it is important to know that the work is being conducted using the correct reagents. This is no less important for cell cultures, since if cell cultures are not what they are reported to be then work can be invalidated and resources wasted. There is now considerable evidence of gross cross-contamination of cell lines, in particular with HeLa (Prod. No. 93021013-1v1) where up to 16 lines was offered to ECACC with DNA profiles identical to HeLa. These include Hep 2,WISH, INT 407, Chang liver and Giradi heart. To minimize the risk of working with contaminated cell lines it is advisable to obtain cells from a recognized source such as a culture collection that will have confirmed the identity of the cells as part of the banking process. Tests used to authenticate cell cultures include iso-enzyme analysis, karyotyping/ cytogenetic analysis and more recently molecular techniques of DNA profiling. Whilst most of the techniques above are generalized tests and are applicable to all cell lines additional specific tests may also be required to confirm the presence of a product or antigen of interest.

        Iso-Enzyme Analysis

        Iso-enzymes are a series of enzymes present in different species that have similar catalytic properties but differ in their structure. By studying the iso-enzymes present in cell lines it is possible to identify the species from which the cell line was derived. The technique is also used as a means of excluding the possibility of gross cross-contamination of the cell line with another culture of a different species.

        The principles upon which iso-enzyme analysis is based are:

        Each iso-enzyme has multiple gene loci coding for different polypeptides with identical enzyme activity (e.g. lactate dehydrogenase, LD)

        Electrophoretic migration rates change dependent on sub-unit composition e.g. LD has five possible iso-forms (LD 1-5)

        Different species have different combinations of these iso-forms

        Using a typical panel of 4 iso-enzymes a composite picture is built up enabling the species of origin to be determined by the use of reference tables

        DNA Fingerprinting

        DNA fingerprinting enables the following:

        Identification of individual cell lines from the same species

        Confirmation of the identity of cell banks compared to reference master stocks

        Detection of cross-contamination

        Multi locus DNA fingerprinting and multiplex - PCR DNA profiling are the methods used routinely as part of ECACC's routine cell banking procedures.

        Multi Locus DNA Fingerprinting

        Uses multi locus Jeffrey’s probes 33.15 or 33.6, along with Southern blotting technology producing a complex banding pattern.

        Probes cross-hybridize with most common species

        Has the disadvantage that the profiles require visual interpretation and comparison with other samples can be subjective

        Multiplex - PCR (STR) DNA profiling

        Uses a set of primers (9 used at ECACC) recognizing micro-satellites using PCR and automated DNA sequencing techniques

        Primers are species specific and are used only for human cell lines

        Produces a color-coded banding pattern, that translates into a digital code that can easily be stored on a database and compared to other stored profiles

        ad image
        提问
        扫一扫
        丁香实验小程序二维码
        实验小助手
        丁香实验公众号二维码
        扫码领资料
        反馈
        TOP
        打开小程序