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 libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent 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 revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated 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 frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.
Adjaye
J
J
Molecular Embryology Unit, Institute of Child Health, 30 Guilford Street, London, UK. j.adjaye@ich.ucl.ac.uk
Bolton
V
V
Monk
M
M
D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D001324:Autoantigens / Q000235:genetics; D001483:Base Sequence; D001755:Blastocyst / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D018899:CpG Islands / Q000235:genetics; D003599:Cytoskeleton / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005314:Embryonic and Fetal Development / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015723:Gene Library; D005796:Genes / Q000235:genetics; D005801:Genes, Homeobox / Q000235:genetics; D018392:Genomic Imprinting / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007633:Keratins / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000235:genetics; D017231:Proto-Oncogene Proteins c-mos / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017411:Ribonucleoproteins, Small Nuclear; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D055517:snRNP Core Proteins
D000199:Actins; D001324:Autoantigens; D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D017411:Ribonucleoproteins, Small Nuclear; C526417:SNRPN protein, human; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D055517:snRNP Core Proteins; D007633:Keratins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007041:Hypoxanthine Phosphoribosyltransferase; D017231:Proto-Oncogene Proteins c-mos