Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos

We describe an improved highly sensitive method for generating cDNA librari es containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. T he embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. F irst-strand cDNA synthesis was carried out directly on the bound mRNA, foll owed by PCR designed to amplify the cDNA molecules synthesized in their ent irety. The complexities of the libraries are between 10(5) and 10(6) indepe ndent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3. 0 kb. PCR analysis of the embryonic libraries for specific genes has reveal ed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 an d the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the ce ll cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of r andom clones showed the presence of a variety of sequences, such as human c horionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protei n (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TW IST, which encodes a basic helix-loop-helix (bHLH) transcription factor imp licated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a freq uency of 16% of randomly picked clones. The libraries are a valuable resour ce, providing longer cDNAs representing genes expressed during human preimp lantation development. (C) 1999 Elsevier Science B.V. All rights reserved.