Isolation of stem cell-specific cDNAs from hematopoietic stem cell populations.

We have begun to isolate gene sequences that are specifically expressed in hematopoietic stem cells (HSCs). There are at least three fundamental requirements for the isolation of HSC-specific transcripts. First, highly enriched populations of HSCs, and an HSC-depleted cell population for comparison must be isolated. Secondly, the gene isolation procedures must be adapted to accommodate the small amounts of RNA obtained from purified HSCs. Finally, a defined screening strategy must be developed to focus on sequences to be examined in more detail. In this report, we describe the characterization of populations of HSCs that are highly enriched (Lin- c-kitHI) or depleted (Lin- c-kitNEG) of HSCs. We compared two methods for gene isolation, differential display polymerase chain reaction (DD-PCR) and subtractive hybridization (SH), and found that the latter was more powerful and efficient in our hands. Lastly we describe the strategy that we have developed to screen clones for further study.

Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene.

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-based method, requires many primer sets and amplification reactions to scan the entire protein-coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.

Ovulated oocytes collected from PMSG/hCG-treated and cycling Djungarian or Siberian hamsters (Phodopus sungorus) are structurally similar with no evidence of polar body formation, indicating arrest in meiosis I.

This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4-5 weeks; n = 70) received 5 IU PMSG followed 50-52 hr later by 10 IU hCG. Ovulated oocytes were collected 14-20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7-12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 +/- 0.4 (range 1-7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 +/- 0.8 (range 0-22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)