Subtractive cDNA cloning using oligo(dt)₃₀-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells

Abstract

The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-treated NEC14 cells using an oligo(dT)30 primer covalently linked to Latex particles. After removal of the mRNA template by heat-denaturation and centrifugation, the subtractive hybridization was carried out between the cDNA-oligo(dT)30-Latex and mRNA from untreated NEC14 cells. Unhybridized mRNA collected by centrifugation was hybridized repeatedly to the cDNA-oligo(dT)30-Latex and subtractive mRNA was converted to cDNA. The subtractive cDNA was then amplified by PCR and cloned into pBluescript II KS-. The cDNA library thus constructed consisted of approximately 10,000 independent clones with cDNA inserts of 1.7 Kb on average. Differential hybridization of these transformants indicated that approximately 3% of them contained cDNA inserts specific to the undifferentiated EC cells, some of which were derived from low abundance mRNAs.

Id proteins in cell cycle control and cellular senescence

“…In addition to this ability to inhibit DNA binding, the name Id is often taken to re¯ect the potential role of these proteins in inhibiting di erentiation. For example, the expression of Id genes is down-regulated upon di erentiation in many cell types (Benezra et al, 1990;Hara et al, 1991a;Sun et al, 1991;Kreider et al, 1992;Le Jossic et al, 1994;Einarson and Chao, 1995). Conversely, ectopic expression of Id1 inhibits B-cell development (Sun, 1994) and the di erentiation of muscle (Jen et al, 1992), myeloid (Kreider et al, 1992), erythroid (Shoji et al, 1994;Lister et al, 1995) and mammary epithelial cells (Desprez et al, 1995(Desprez et al, , 1998.…”

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

“…Although the theory of differential display is very simple, it costs too much time and the false positive rate is quite high [24], [25]. Traditional subtractive hybridization methods required several rounds of hybridization and are not well suited for the identification of rare messages [26][27][28] . Based on the applying of selective amplification of differentially expressed sequences, besides overcoming technical limitation of traditional subtraction methods [29], [30], suppression subtractive hybridization leads to enrichment specific-expression library.…”

Functional Genomics and Proteomics: Application in Neurosciences

“…Initial approaches to differential gene expression include subtractive cloning, 8 serial analysis of gene expression (SAGE), 9 and subtractive hybridisation techniques. 10 Differential display is a less laborious and more widely used approach, 11 and has yielded promising results, particularly with an adapted indexing based differential display PCR (DD-PCR) technique.…”