The necessity of identity assessment of animal intestinal cell lines: A case report - PubMed (original) (raw)

The necessity of identity assessment of animal intestinal cell lines: A case report

Klaus G Steube et al. Cytotechnology. 2012 Aug.

Abstract

Eight intestinal cell lines, established from different animal species were submitted to DSMZ (German Collection of Microorganisms and Cell Cultures) in order to analyze their species of origin and their microbial contamination. Species identity was determined by PCR targeting mitochondrial genes and hence confirmed by sequencing the amplified PCR products. For three cell lines (CIEB, CLAB, PSI-1) we confirmed the species identity, whereas the species of origin of the three other cell lines (B6, B10XI and IPEC) was not the expected one: B6 and B10XI cells, which were supposed to be of chicken origin were identified as porcine cells. IPEC, allegedly a sub clone of the well-known porcine intestinal cell line IPEC-J2, was of bovine instead of porcine origin. However, two further IPEC-clones, namely IPEC-1 and IPEC-J2, provided by another source were shown to be derived from the correct species (i.e. pig). Furthermore, six out of these eight cell lines turned out to be highly contaminated with mycoplasma. Alerted by this high incidence of infected and false specified cell lines, we feel obliged to inform all those working with animal intestinal cell lines and we strongly recommend verifying the species identity before using them. Also, the presence of mycoplasma should be tested when taking the cells in culture for the first time, and this mycoplasma control should be repeated at regular time intervals (e.g. every 4 weeks).

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Figures

Fig. 1

Fig. 1

Analysis of genomic DNA from different cell lines using bovine (a) or porcine primer pairs (b). Amplified DNA fragments were detected after ethidium bromide staining of 1.2% agarose gels. DNA molecular weight markers of 500 bp and 1,000 bp are bold. a, primer pair: Mito-Bov. Lane 1: CLAB; lane 2: CIEB; lane 3: PSI-1; lane 4: IPEC; lane 5: PK-15 (pig, DSMZ ACC 640); lane 6: GM-7373 (bovine, DSMZ ACC 109); lane 7: water control (no DNA). Expected fragment size: 271 bp. b, primer pair: Mito-Sus. Lane 1: CLAB; lane 2: HELA (human, DSMZ ACC 57); lane 3: CIEB; lane 4: NIH-3T3 (mouse, DSMZ ACC 59); lane 5: PSI-1; lane 6: PC-12 (rat, DSMZ ACC 159); lane 7: IPEC; lane 8: PK-15 (pig, DSMZ ACC 640); lane 9: LAT (ovine, DSMZ ACC 349); lane 10: LLC-PK1 (pig, DSMZ ACC 637); lane 11: BHK-21 (hamster, DSMZ ACC 61); lane 12: water control (no DNA). Expected fragment size: 532 bp

Fig. 2

Fig. 2

Analysis of genomic DNA from different cell lines using a duplex-PCR with dog and chicken primer pairs. Amplified DNA fragments were detected after ethidium bromide staining of 1.2% agarose gels. DNA molecular weight marker of 500 bp is bold. Lane 1: B6; lane 2: B10XI; lane 3: MDCK (dog); lane 4: HELA (human, DSMZ ACC 57); lane 5: MDCK (dog); lane 6: NIH-3T3 (mouse, DSMZ ACC 59); lane 7: DT-40 (chicken, DSMZ ACC 636); lane 8: MDBK (bovine, DSMZ ACC 174); lane 9: HD-11 (chicken); lane 10: EBL (bovine, DSMZ ACC 192); lane 11: LMH-2 (chicken); lane 12: water (no DNA). Expected fragment size for dog: 154 bp and chicken: 474 bp

Fig. 3

Fig. 3

Analysis of genomic DNA from different cell lines using a multiplex-PCR with bovine, horse, ovine and porcine primer pairs. Amplified DNA fragments were detected after ethidium bromide staining of 1.2% agarose gels. DNA molecular weight markers of 500 and 1,000 bp are bold. Lane 1: IPEC-J2; lane 2: IPEC; lane 3: LAT (ovine, DSMZ ACC 349); lane 4: reference DNA horse; lane 5: IPEC-1; lane 6: CIEB (bovine); lane 7: FLK-BLV-044 (ovine, DSMZ ACC 153); lane 8: reference DNA donkey; lane 9: DT-40 (chicken, DSMZ ACC 636); lane 10: B6; lane 11: B10XI; lane 12: water (no DNA). Expected fragment sizes: porcine, 691 bp; bovine 415 bp; ovine 300 bp; horsel/donkey, 244 bp

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