Development of a panel of monochromosomal somatic cell hybrids for rapid gene mapping

Kelsell, David P.; Rooke, Lesley; Warne, D.; Bouzyk, Mark; Cullin, L.; Cox, S A; West, L. F.; Povey, S.; Spurr, N.K.

doi:10.1111/j.1469-1809.1995.tb00743.x

Cited by 78 publications

(41 citation statements)

References 29 publications

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“…This sequence was used to design insertspecific primer 1 (ISP1) and ISP2 that would generate insert-specific PCR products ( Figure 3). These were used with DNA from a monochromosomal rodent-human somatic cell hybrid panel (25), and the insert-specific sequence was shown to originate from human chromosome 2 ( Figure 3). In addition, a third ISP, ISP3, was used in conjunction with X chromosome-specific primer R (XSPR) to generate a hybrid PCR product that was specific for the HPT telomeric boundary (Figure 3).…”

Section: Figurementioning

confidence: 99%

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism

Bowl¹,

Nesbit²,

Harding³

et al. 2005

J. Clin. Invest.

135

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Section: Figurementioning

confidence: 99%

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism

Bowl¹,

Nesbit²,

Harding³

et al. 2005

J. Clin. Invest.

135

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“…Two PCR primers (gmp1 and gmp2) were designed to bind to this unknown 84 bp sequence, and PCR was performed on a human monochromosomal somatic cell hybrid DNA panel. 29 A single band of the expected size was obtained from human positive control DNA and from the hybrid containing human chromosome 22, but not from mouse and hamster negative control DNA or from any of the other hybrids containing chromosomes 1-21, X and Y (data not shown). Consistent with this result, a DNA database homology search revealed that the 5Ј 84 bp sequence obtained from the 1.8 kb patient 2 product matched 100% to a chromosome 22 PAC clone (DJ130H16) which has recently been sequenced by the St Louis Genome Sequencing Center.…”

Section: Analysis Of a Novel T(14;22) Breakpoint Isolated From A Primmentioning

confidence: 99%

“…Each 50 l PCR reaction contained 200 m dNTPs, 60 mm Tris-SO 4 (pH 9.1), 18 mm (NH 4 ) 2 SO 4 2 mm MgSO 4 , 12.5 pmoles each primer, 100 ng genomic DNA, and 1 l Elongase. Amplification of genomic DNA sample (100 ng/reaction) or a human monochromosomal somatic cell hybrid DNA panel (40 ng/reaction) 29 was performed using primers gmp0-4 as described above but using 30 cycles of the following conditions: 95°C for 30 s, 56°C for 30 s, and 68°C for 30 s.…”

Section: Standard Dna Amplificationsmentioning

confidence: 99%

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1999

Leukemia

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Immunoglobulin class switching occurs as a result of recombination between pairs of switch region sequences located 5Ј to each constant heavy chain gene except C␦. In the B cell neoplasm multiple myeloma, tumour cells have generally undergone class switching and often contain oncogenic sequences translocated into switch regions, presumably as a result of aberrant switch recombination. We have developed a method (LDV-PCR) which combines long distance PCR with one-sided vectorette PCR that is capable of detecting and isolating both normal and aberrant switch recombination breakpoints from multiple myeloma cell lines and primary multiple myeloma tumour material. Using LDV-PCR we have directly cloned the translocation breakpoints present in two multiple myeloma cell lines and isolated a normal productive switch recombination event from a primary tumour. Furthermore, we have isolated a novel translocation t(14;22)(q32;q12) from a primary tumour sample and have demonstrated that internal deletions within switch regions can occur in multiple myeloma cells. Compared to a Southern blotting approach, LDV-PCR is simpler and more rapid to perform, allows the simultaneous detection and isolation of recombination events, and can also be applied to amounts of DNA which are too low to permit the conventional cloning of recombination breakpoints.

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“…Establishment of somatic hybrid cells containing t(5;14)-SRIK-NKL cells were fused with TK-minus mouse 3T3 mutant cell line using standard PEG protocols [30][31][32][33]. The HAT-resistant cells were subcloned and propagated for DNA/RNA extraction, SKY/FISH analysis and molecular mapping.…”

Section: Establishment and Maintenance Of Srik-nkl Cell Linementioning

confidence: 99%

Genomic analysis of CD8+ NK/T cell line, ‘SRIK-NKL’, with array-based CGH (aCGH), SKY/FISH and molecular mapping

et al. 2008

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We performed aCGH, SKY /FISH, molecular mapping and expression analyses on a permanent CD8+ NK/T cell line, 'SRIK-NKL' established from a lymphoma (ALL) patient, in attempt to define the fundamental genetic profile of its unique NK phenotypes. aCGH revealed hemizygous deletion of 6p containing genes responsible for hematopoietic functions. The SKY demonstrated that a constitutive reciprocal translocation, rcpt(5;14)(p13.2;q11) is a stable marker. Using somatic hybrids containing der(5) derived from SRIK-NKL, we found that the breakpoint in one homologue of no. 5 is located upstream of IL7R and also that the breakpoint in no. 14 is located within TRA@. The FISH analysis using BAC which contains TRA@ and its flanking region further revealed a ~231 kb deletion within 14q11 in the der(5) but not in the normal homologue of no. 14. The RT-PCR analysis detected mRNA for TRA@ transcripts which were extending across, but not including, the deleted region. IL7R was detected at least at mRNA levels. These findings were consistent with the immunological findings that TRA@ and IL7R are both expressed at mRNA levels and TRA@ at cytoplasmic protein levels in SRIK-NKL cells. In addition to rept(5;14), aCGH identified novel copy number abnormalities suggesting that the unique phenotype of the SRIK-NKL cell line is not solely due to the TRA@ rearrangement. These findings provide supportive evidence for the notion that SRIK-NKL cells may be useful for studying not only the function of NK cells but also genetic deregulations associated with leukemiogenesis.

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Development of a panel of monochromosomal somatic cell hybrids for rapid gene mapping

Cited by 78 publications

References 29 publications

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism

An interstitial deletion-insertion involving chromosomes 2p25.3 and Xq27.1, near SOX3, causes X-linked recessive hypoparathyroidism

True

Genomic analysis of CD8+ NK/T cell line, ‘SRIK-NKL’, with array-based CGH (aCGH), SKY/FISH and molecular mapping

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