Construction of human embryonic cDNA libraries: HD, PKD1 and BRCA1 are transcribed widely during embryogenesis

Abstract

We used the polymerase chain reaction (PCR) to construct cDNA libraries from small amounts of tissue and to screen cDNA libraries efficiently for the presence of given sequences. To isolate genes expressed in early human development, we constructed both oligo dT-primed and random hexamer-primed cDNA libraries from ten different tissues of human embryos aged 53 to 78 days post conception. Given the small amount of RNA available, it was necessary to amplify the resultant cDNA using PCR to generate sufficient amounts of cDNA for library construction. As a result of using PCR followed by sizing to eliminate smaller synthesized fragments, the size of the synthesized product was ≧ 650 base pairs and the average initial complexity of the given libraries was 10⁶. We screened these cDNA libraries efficiently using PCR. Primers corresponding to a given gene were used to amplify DNA from phages encompassing a cDNA library. Successful amplification of the appropriate-sized fragment demonstrated that the DNA in question was transcribed in a given tissue. We demonstrated that HD (huntingtin, the protein transcribed from the Huntington disease locus), PKD1 (the most common gene responsible for familial polycystic kidney disease) and BRCA1 (a gene responsible for familial breast cancer) are synthesized nearly ubiquitously (including during embryogenesis). Thus, these human embryonic cDNA libraries constitute a unique resource to study early human development.