Bidirectional transcriptional activity of PGK-neomycin and unexpected embryonic lethality in heterozygote chimeric knockout mice.

In an effort to create a conventional knockout mouse model for multiple endocrine neoplasia type 1 (MEN1), we targeted disruption of the mouse Men1 gene through homologous recombination in ES cells. Men1 exons 2-4 were replaced by a PGK-neomycin cassette inserted in the opposite direction of Men1 transcription (Men1(MSK/+)). Unexpectedly, the Men1 conventional knockout was lethal in heterozygous, chimeric animals. Analysis of embryos revealed late gestational lethality with some embryos showing omphalocele. This was a very surprising phenotype, given that humans and mice that are heterozygotes for loss of function mutations in MEN1 are phenotypically normal except for a risk of endocrine tumors. Northern analysis of Men1(MSK/+) embryonic stem cell RNA revealed the presence of an abundant, novel transcript of 2.1 kb, in addition to the expected wild-type transcripts of 2.7 kb and 3.1 kb. RT-PCR analysis identified this aberrant transcript as arising from the antisense strand of the PGK promoter. We hypothesize that this transcript is producing either a toxic effect at the RNA level, or a dominant negative effect through the production of an amino-terminal truncated protein product. This example serves as a cautionary reminder that mouse knockouts using PGK-neo may sometimes display phenotypes that reflect more than just the loss of function of the targeted gene.

A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.

Oligonucleotide microarray based detection of repetitive sequence changes.

Prior studies of oligonucleotide microarray-based mutational analysis have demonstrated excellent sensitivity and specificity except in circumstances where a frameshift mutation occurs in the context of a short repeated sequence. To further evaluate this circumstance, a series of nucleic acid samples having heterozygous mutations within repetitive BRCA1 sequence tracts was prepared and evaluated. These mutations included single nucleotide insertions and deletions in homopolymer runs, insertions and deletions of trinucleotide repeats, and duplications. Two-color comparative hybridization experiments were used wherein wild type reference and test targets are co-hybridized to microarrays designed to screen the entire BRCA1 coding sequence for all possible sequence changes. Mutations in simulated heterozygote samples were detected by observing relative losses of test target hybridization signal to select perfect match oligonucleotide probes. While heterozygous mutations could be readily distinguished above background noise in 9/19 cases, it was not possible to detect alterations in a poly dA/dT tract, small triplet repeat expansions, and a 10 bp direct repeat. Unexpectedly, samples containing (GAT)(3) triplet repeat expansions showed significantly higher affinity toward specific perfect match probes relative to their wild type counterparts. Therefore, markedly increased as well as decreased test sample hybridization to perfect match probes should be used to raise a suspicion of repetitive sequence changes.

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates.

Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.

Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.

Evolutionary sequence comparisons using high-density oligonucleotide arrays.

We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes.

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays.

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.