Wild-type Crosses Research Articles

One-Pot Era-CRISPR/Cas12a System for Rapid and Sensitive Detection of Multiple NPM1 Mutations

Nucleophosmin 1 (NPM1) gene c.863_864 4-bp insertion mutations occur in 30% of adult acute myeloid leukemia (AML) patients, which is associated with a unique pathological phenotype and good prognostic significance. The detection of NPM1 mutations is important for risk stratification, treatment guidance, and minimal residual disease monitoring in AML. However, the current detection methods have their limitations in terms of equipment requirements, sensitivity, turnaround time, price, etc. Moreover, simultaneous detection of multiple NPM1 mutation types is challenging. Here, we developed a one-pot ERA-CRISPR/Cas12a-based assay in which only one guide crRNA is needed to detect hundreds of NPM1 gene variants, covering over 97% of NPM1-mutated AML cases. By optimizing multiple parameters, this assay finally achieved a sensitivity of as low as 0.01% within 30 min detection under constant 39℃, without wild-type related cross signal. In clinical detection, 21 NPM1 mutation positive cases from 70 AML patient were successfully screened out using our method, including two cases not detected by Sanger sequencing. This method is one-step, rapid, sensitive, specific, inexpensive, and without the need of Sequencer or temperature cycler. It holds the potential to assist clinicians in point of care testing.

QTL mapping of the narrow-branch “Pendula” phenotype in Norway spruce (Picea abies L. Karst.)

Pendula-phenotyped Norway spruce has a potential forestry interest for high-density plantations. This phenotype is believed to be caused by a dominant single mutation. Despite the availability of RAPD markers linked to the trait, the nature of the mutation is yet unknown. We performed a quantitative trait loci (QTL) mapping based on two different progenies of F1 crosses between pendula and normal crowned trees using NGS technologies. Approximately 25% of all gene bearing scaffolds of Picea abies genome assembly v1.0 were mapped to 12 linkage groups and a single QTL, positioned near the center of LG VI, was found in both crosses. The closest probe markers placed on the maps were positioned 0.82 cm and 0.48 cm away from the Pendula marker in two independent pendula-crowned × normal-crowned wild-type crosses, respectively. We have identified genes close to the QTL region with differential mutations on coding regions and discussed their potential role in changing branch architecture.

RETRACTED ARTICLE: BrRNE cleaves RNA in chloroplasts, regulating retrograde signals in Brassica rapa L. ssp. pekinensis

Brassica rapa RNE participates in the processing of polycistronic precursor transcripts into mature monocistronic mRNAs in plastids, thereby sending strong retrograde signals. Leaf color is one of the most important agronomic traits for Chinese cabbage. Not only is it closely linked to photosynthesis, thereby affecting plant growth, but it also influences consumer preference in the marketplace. A pale-green mutant rne was produced by EMS mutagenesis of Chinese cabbage inbred line A03. Chlorophyll content, photosynthetic rate, actual quantum efficiency (φPSII), and maximum quantum efficiency (Fv/Fm) of photosystem II (PSII) were all reduced in rne plants. Genetic analysis indicated that the pale-green trait was controlled by a pair of recessive alleles. Using mixed pool sequencing of F2 individuals derived from an rne × wild-type cross, we identified the essential gene Brassica rapa RNase E (BrRNE), which is responsible for chloroplast development. BrRNE cleaves polycistronic RNA in Chinese cabbage A03 plastids, but rne plants are defective in RNA processing and show reduced translation levels of the seven plastid genes, BrpsaB, BrpsaA, BrpsbA, BrpsbD, BrpsbB, BrpetA, and Brycf4. Abnormal RNA processing in the plastids sends retrograde signals that markedly regulate the expression of nuclear genes, upregulating genes that participate in ribosome and DNA replication pathways and repressing photosynthesis-associated nuclear genes (PhANGs). Our study reveals a new regulatory mechanism by which plastid RNA cleavage influences plastid development and leaf color, sending retrograde signals that affect the expression of nuclear genes in Brassica.

Effects of Grape NAC Transcription Factor DRL1 on Floral Organogenesis in Tobacco

Grape (Vitis vinifera) is among the world’s most important fruit crops and is a commonly used woody plant for genomics and post-genomics research. NAC transcription factors play central roles in plant growth and development, floral organ morphogenesis, and responses to biological stress. It is therefore important to identify key transcription factors from grape and clarify their mechanisms of action to generate genetic resources for grape molecular improvement. Our research group previously cloned a NAC transcription factor from V. vinifera ‘Yatomi Rosa’ [drought and leaf roll gene 1 (DRL1)] and demonstrated that it caused dwarfing of tobacco (Nicotiana benthamiana) plants when overexpressed. In the present work, we demonstrate that overexpression of DRL1 in transgenic tobacco delays flowering time and markedly reduces pollen viability. Furthermore, crosses between male DRL1 transgenic tobacco and female wild-type tobacco exhibit substantially lower fruit set, fruit and seed weights, fruit and seed shape indices, and seed germination rates than selfed wild-type plants or crosses with a transgenic female parent. DLR1 overexpression strongly influences flowering time and reproduction in transgenic tobacco, primarily through its effects on pollen development. These results provide a foundation for further functional characterization of DLR1 in grape.

Sexual Differentiation Is Coordinately Regulated by Cryptococcus neoformans CRK1 and GAT1.

The heterothallic basidiomycetous fungus Cryptococcus neoformans has two mating types, MATa and MATα. Morphological progression of bisexual reproduction in C. neoformans is as follows: yeast to hyphal transition, filament extension, basidium formation, meiosis, and sporulation. C. neoformans Cdk-related kinase 1 (CRK1) is a negative regulator of bisexual mating. In this study, we characterized the morphological features of mating structures in the crk1 mutant and determined the genetic interaction of CRK1 in the regulatory networks of sexual differentiation. In the bilateral crk1 mutant cross, despite shorter length of filaments than in the wild-type cross, dikaryotic filaments and other structures still remained intact during bisexual mating, but the timing of basidium formation was approximately 18 h earlier than in the cross between wild type strains. Furthermore, gene expression analyses revealed that CRK1 modulated the expression of genes involved in the progression of hyphal elongation, basidium formation, karyogamy and meiosis. Phenotypic results showed that, although deletion of C. neoformans CRK1 gene increased the efficiency of bisexual mating, filamentation in the crk1 mutant was blocked by MAT2 or ZNF2 mutation. A bioinformatics survey predicted the C. neoformans GATA transcriptional factor Gat1 as a potential substrate of Crk1 kinase. Our genetic and phenotypic findings revealed that C. neoformans GAT1 and CRK1 formed a regulatory circuit to negatively regulate MAT2 to control filamentation progression and transition during bisexual mating.

The complex underpinnings of genetic background effects

Genetic interactions between mutations and standing polymorphisms can cause mutations to show distinct phenotypic effects in different individuals. To characterize the genetic architecture of these so-called background effects, we genotype 1411 wild-type and mutant yeast cross progeny and measure their growth in 10 environments. Using these data, we map 1086 interactions between segregating loci and 7 different gene knockouts. Each knockout exhibits between 73 and 543 interactions, with 89% of all interactions involving higher-order epistasis between a knockout and multiple loci. Identified loci interact with as few as one knockout and as many as all seven knockouts. In mutants, loci interacting with fewer and more knockouts tend to show enhanced and reduced phenotypic effects, respectively. Cross–environment analysis reveals that most interactions between the knockouts and segregating loci also involve the environment. These results illustrate the complicated interactions between mutations, standing polymorphisms, and the environment that cause background effects.

Development, evaluation and genetic analysis of sulfosulfuron herbicide resistance in sorghum

Herbicide tolerant varieties in combination with herbicide seed treatments can be used to manage Striga. However, there are no herbicide resistant sorghum varieties in Kenya. The objectives of this study, therefore, were to develop sulfosulfuron resistance in sorghum, to determine the level of resistance in resultant herbicide tolerant mutants, and to determine the genetic inheritance of herbicide tolerance in sorghum. Five ethyl methane sulphonate (EMS)-derived sulfosulfuron tolerant mutants (designated hb6, hb8, hb12, hb56, and hb462) were identified and selfed to M4 generation. Varying rates of sulfosulfuron, either as a spray or seed coat, were applied to determine the level of tolerance of the mutant lines. Mutant lines were also crossed with the wild-type Seredo and among themselves to determine mode of inheritance. Results showed that the susceptible wild-type Seredo was killed at the lowest herbicide rates of 0.5 g ha-1 and 1 g ha-1 sulfosulfuron. Dry matter from the spraying and seed coating experiments showed mutants to be up to 170 times more resistant to sulfosulfuron than the wild-type. The LD50 values indicated a general trend of hb46 > hb12 > hb462 ~ hb56 > hb8 for level of tolerance under both spraying and seed coating experiments. The F2 progeny of mutant X wild-type crosses segregated in a 1:2:1 fashion for resistant, intermediate, and susceptible, indicating semi-dominant inheritance. Intercrosses between mutant lines did not segregate for resistance in the F2 generation indicating the same mutation could be responsible for the tolerance in all five mutants.

Murine red blood cells from genetically distinct donors cross-regulate when stored together.

Donor variability of red blood cell (RBC) storage has been observed in both humans and animal models. We utilized a strain of mice with RBCs known to store well (B6) and a strain known to store poorly (FVB) to test the hypothesis that RBCs affected the storage of other RBCs. Five strains of mice were used: 1) transgenic B6 mice expressing green fluorescent protein (GFP) in their RBCs (GFP.B6), 2) wild-type B6 mice, 3) wild-type FVB mice, 4) F1 crosses between GFP.B6 and FVB mice (GFP.F1), and 5) the analogous wild-type (B6xFVB) F1 cross. GFP.B6 or GFP.F1 RBCs were mixed with wild-type (non-GFP) RBCs from B6 or FVB strains before storage. Twenty-four-hour RBC recoveries were determined for stored RBCs by enumerating circulating GFP+ RBCs by flow cytometry. Twenty-four-hour recoveries of GFP.F1 RBCs was increased by co-storage with B6 RBCs but decreased by co-storage with FVB RBCs. This effect was dose dependent when tested with GFP.B6 RBCs; the more FVB blood added, the worse the 24-hour recoveries became. RBC cross-regulation did not occur when B6 and FVB RBCs were separated by a semipermeable membrane with a 0.4-µm size cutoff. These findings demonstrate that RBCs affect the storage of other RBCs, in both positive and negative directions, indicating not only that RBC storage is intrinsic to the RBC but that RBC-RBC communication occurs. Additional studies will be required to determine the nature of this effect and if these findings translate into human RBC storage.

Revisiting Suppression of Interspecies Hybrid Male Lethality in Caenorhabditis Nematodes.

Within the nematode genus Caenorhabditis, Caenorhabditis briggsae and C. nigoni are among the most closely related species known. They differ in sexual mode, with C. nigoni retaining the ancestral XO male–XX female outcrossing system, while C. briggsae recently evolved self-fertility and an XX-biased sex ratio. Wild-type C. briggsae and C. nigoni can produce fertile hybrid XX female progeny, but XO progeny are either 100% inviable (when C. briggsae is the mother) or viable but sterile (when C. nigoni is the mother). A recent study provided evidence suggesting that loss of the Cbr-him-8 meiotic regulator in C. briggsae hermaphrodites allowed them to produce viable and fertile hybrid XO male progeny when mated to C. nigoni. Because such males would be useful for a variety of genetic experiments, we sought to verify this result. Preliminary crosses with wild-type C. briggsae hermaphrodites occasionally produced fertile males, but they could not be confirmed to be interspecies hybrids. Using an RNA interference (RNAi) protocol that eliminates any possibility of self-progeny in Cbr-him-8 hermaphrodites, we found sterile males bearing the C. nigoni X chromosome, but no fertile males bearing the C. briggsae X, as in wild-type crosses. Our results suggest that the apparent rescue of XO hybrid viability and fertility is due to incomplete purging of self-sperm prior to mating.

Double-strand break repair and genetic recombination in topoisomerase and primase mutants of bacteriophage T4.

The effects of primase and topoisomerase II deficiency on the double-strand break (DSB) repair and genetic recombination in bacteriophage T4 were studied in vivo using focused recombination. Site-specific DSBs were induced by SegC endonuclease in the rIIB gene of one of the parents. The frequency/distance relationship was determined in crosses of the wild-type phage, topoisomerase II mutant amN116 (gene 39), and primase mutant E219 (gene 61). Ordinary two-factor (i×j) and three-factor (i k×j) crosses between point rII mutations were also performed. These data provide information about the frequency and distance distribution of the single-exchange (splice) and double-exchange (patch) events. In two-factor crosses ets1×i, the topoisomerase and primase mutants had similar recombinant frequencies in crosses at ets1-i distances longer than 1000 bp, comprising about 80% of the corresponding wild-type values. They, however, differ remarkably in crosses at shorter distances. In the primase mutant, the recombinant frequencies are similar to those in the wild-type crosses at distances less than 100 bp, being a bit diminished at longer distances. In two-factor crosses ets1×i of the topoisomerase mutant, the recombinant frequencies were reduced ten-fold at the shortest distances. In three-factor crosses a6 ets1×i, where we measure patch-related recombination, the primase mutant was quite proficient across the entire range of distances. The topoisomerase mutant crosses demonstrated virtually complete absence of rII(+) recombinants at distances up to 33 bp, with the frequencies increasing steadily at longer distances. The data were interpreted as follows. The primase mutant is fully recombination-proficient. An obvious difference from the wild-type state is some shortage of EndoVII function leading to prolonged existence of HJs and thus stretched out ds-branch migration. This is also true for the topoisomerase mutant. However, the latter is deficient in the ss-branch migration step of the DSB repair pathway and partially deficient in HJ initiation. In apparent contradiction to their effects on the DSB-induced site-specific recombination, the topoisomerase and primase mutants demonstrated about 3-8-fold increase in the recombinant frequencies in the ordinary crosses, with the recombination running exclusively via patches. This implies that most of the spontaneous recombination events are not initiated by dsDNA ends in these mutants.

Development of two sequence-specific PCR markers linked to the le gene that reduces pod shattering in narrow-leafed Lupin (Lupinus angustifolius L.)

Wild types of narrow-leaf lupin (Lupinus angustifolius L.) have seed pods that shatter upon maturity, leading to the loss of their seeds before or during the harvest process. Two recessive genes have been incorporated into domesticated cultivars of this species to maximize harvest-ability of the produce. One of these genes is called lentus (le). Two microsatellite - anchored fragment length polymorphism (MFLP) candidate markers were identified as closely linked to the le gene in a recombinant inbred line (RIL) population derived from a domesticated x wild type cross. The candidate MFLP markers were isolated from the gel, re-amplified by PCR, cloned and sequenced. The MFLP polymorphisms were converted into sequence-specific PCR-based markers. Linkage analysis by MapManager indicated that one of the markers, LeM1, was 2.6 centiMorgans (cM) and the other, LeM2, was 1.3 cM from the gene, with both being on the same side. The correlation between the marker genotype and the plant phenotype for the le gene is 95% for the Australian cultivars, and approximately 36% on wild types tested. These markers may be useful in marker assisted selection for the le gene when introgressing wild material into lupin breeding programs.

Relative Activity of a Tobacco Hybrid Expressing High Levels of a Tobacco Anionic Peroxidase and Maize Ribosome-Inactivating Protein against Helicoverpa zea and Lasioderma serricorne

Tobacco (Nicotiana tabacum) plants grown from seed obtained by crossing a tobacco line that expressed an activated maize ribosome-inactivating protein (RIP) with a line that overexpressed tobacco anionic peroxidase were tested for their effects on corn earworm Helicoverpa zea and cigarette beetle Lasioderma serricorne larvae as compared to the wild-type plant cross. Significant feeding reductions were noted for transgenic plants expressing both resistance proteins as compared to wild-type plants for both H. zea and L. serricorne. Significant increases in mortality were also noted for those insects fed on the transgenic cross as compared to wild-type plants in some cases. Levels of both peroxidase and maize RIP were significantly higher in transgenic as compared to wild-type plants (which did not produce maize RIP). The degree of feeding was significantly negatively correlated with the level of RIP or peroxidase individually.

P0567 THE HEDGEHOG TRANSCRIPTION FACTOR GLI3 MEDIATES THE DEVELOPMENT OF INTESTINAL METAPLASIA

Introduction: Helicobacter pylori infects approximately 50% of children worldwide. Chronic infection can initiate a cascade of pathologic changes within the stomach initially with chronic gastritis and atrophy to intestinal metaplasia (IM). Subsequently, in many cases of IM, carcinoma ensues. The pathobiology of IM, however, is not well understood. Recently, Hedgehog signaling molecules have been identified in both human digestive tract tumours as well as in cell lines derived from digestive tract carcinoma. In the embryo Sonic hedgehog (Shh) contributes to the proper specification of the glandular stomach. In its absence, intestinal transformation of the stomach occurs. The aim of this study was to determine the role of the Hedgehog signaling pathway in the development of IM. Methods: We examined mice mutant in Sonic hedgehog, and the downstream transcription factors, Gli2 and Gli3. All three homozygous mutant mice die at or just before birth. Therefore, pregnant mice from wildtype and heterozygous crosses of the three mutant strains were sacrificed at embryonic day 18.5 and their stomachs were dissected out, fixed in paraformaldehyde, dehydrated and embedded in paraffin. Sections were cut and examined for alkaline phosphatase staining (AP). Immunostaining was performed for the intestinal transcription factor, Cdx2, and intestinal mucin, Muc2, two known markers for IM. Results: The stomachs of both Shh and Gli3 but not Gli2 mutant mice demonstrated positive staining for AP in the distal hindstomach. In the Gli3 but not Shh mutant stomachs, both Cdx2 and Muc2 were found expressed in the distal hindstomach. Gli2 mutant stomachs were also negative for Cdx2 and Muc2 expression. Conclusion: The absence of the activated transcription factor Gli3 leads to the development of intestinal metaplasia in the glandular stomach of the mouse. Concurrently, the transcription factor Cdx2 along with Muc2, which Cdx2 is known to regulate, are actively expressed. However, the presence of the cleaved form of Gli3, as in Shh mutant stomach, prevents this activation. We conclude, therefore, that both forms of Gli3 participate in defining the identity of the gastric gland. Further study of the Hedgehog signalling pathway is likely to improve our understanding of the pathogenesis of intestinal metaplasia.

Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina.

Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR.

Meiotic silencing by unpaired DNA: properties, regulation and suppression.

In Neurospora, a gene not paired with a homolog in prophase I of meiosis generates a signal that transiently silences all sequences homologous to it by a process called meiotic silencing by unpaired DNA (MSUD). Thus a deletion mutation in a heterozygous cross is formally "ascus-dominant" because its unpaired wild-type partner silences itself. We describe in detail the isolation of a mutation, Sad-1(UV), that suppresses the dominance of various ascus-dominant mutations. Additional dominant, semidominant, and recessive Sad-1 alleles have been generated by RIP; the DNA of the dominant RIP alleles becomes methylated, but dim-2-dependent methylation is not necessary for silencing. The barrenness of homozygous Sad-1 crosses is not due to the failure to silence unpaired mating-type mat A-2 mat A-3 genes. Transcripts of sad-1(+) can be detected during the sexual phase in a homozygous wild-type cross, indicating that the gene is expressed even if all DNA can pair normally. Meiotic silencing is confined to the ascus in which DNA is unpaired, and silencing does not spread to neighboring asci in a fruiting body of mixed genetic constitution.

Mutant Male Chlamydomonas reinhardtii Cells with Few Chloroplast Nucleoids and Exceptional Chloroplast Gene Transmission

(male) mutant cell of Chlamydomonas reinhardtii was isolated. It was reduced in size and showed few chloroplast (cp) nucleoids. When smaller mutant cells, obtained through a 3 μm pore filter, were crossed with mt + wild type cells, the frequency of transmission of cp genes was not different from the wild type cross. The cell size and the number of cp nucleoids appear to have no effect on the transmission of cp genes.

Defective splicing of the first nad4 intron is associated with lack of several complex I subunits in the Nicotiana sylvestris NMS1 nuclear mutant.

In this work, we provide evidence for the existence of a nuclear factor involved in the splicing of a specific mitochondrial intron in higher plants. In the Nicotiana sylvestris nuclear NMS1 mutant, defective in both vegetative and reproductive development, the first intron of the nad4 transcript encoding the complex I NAD4 subunit is not removed, whatever the tissue analysed. Transcript patterns of other standard mitochondrial genes are not affected in NMS1. However, numerous polypeptides are missing in two-dimensional in organelle mitochondrial protein synthesis patterns and several nuclear and mitochondrial complex I subunits are present in trace amounts. This indicates that translational or post-translational steps in the synthesis of other mitochondrial proteins are affected. All of these defects co-segregated with the abnormal phenotype in the offspring of a NMS1 x wild-type cross, showing that they are controlled by the same nuclear gene (MS1) or tightly linked loci. Such a complex situation has been described in chloroplasts and mitochondria of fungi, but never in higher plant mitochondria.

Sperm competition in the absence of fertilization in Caenorhabditis elegans.

Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.

Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase.

Recent studies have shown that the p70(s6k)/p85(s6k) signaling pathway plays a critical role in cell growth by modulating the translation of a family of mRNAs termed 5'TOPs, which encode components of the protein synthetic apparatus. Here we demonstrate that homozygous disruption of the p70(s6k)/p85(s6k) gene does not affect viability or fertility of mice, but that it has a significant effect on animal growth, especially during embryogenesis. Surprisingly, S6 phosphorylation in liver or in fibroblasts from p70(s6k)/p85(s6k)-deficient mice proceeds normally in response to mitogen stimulation. Furthermore, serum-induced S6 phosphorylation and translational up-regulation of 5'TOP mRNAs were equally sensitive to the inhibitory effects of rapamycin in mouse embryo fibroblasts derived from p70(s6k)/p85(s6k)-deficient and wild-type mice. A search of public databases identified a novel p70(s6k)/p85(s6k) homolog which contains the same regulatory motifs and phosphorylation sites known to control kinase activity. This newly identified gene product, termed S6K2, is ubiquitously expressed and displays both mitogen-dependent and rapamycin-sensitive S6 kinase activity. More striking, in p70(s6k)/p85(s6k)-deficient mice, the S6K2 gene is up-regulated in all tissues examined, especially in thymus, a main target of rapamycin action. The finding of a new S6 kinase gene, which can partly compensate for p70(s6k)/p85(s6k) function, underscores the importance of S6K function in cell growth.

Nuclear localization signals enhance germline transmission of a transgene in zebrafish.

We report that cytoplasmic injection into zebrafish eggs of 10(4) copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 10(6) copies of naked DNA, increased nuclear uptake of transgene DNA early during embryo development and enhanced transgene integration frequency into the germline of founders. Monitoring the dynamics of nuclear uptake of DNA-NLS complexes by fluorescence in situ hybridization (FISH) of interphase nuclei indicates that NLS enhances both the proportion of nuclei importing DNA during early embryo development, and the amount of DNA imported by individual nuclei. The use of NLS increases the proportion of germline transgenic founders from 14 to 43% (P < 0.01) as assessed by polymerase chain reaction analysis of F1s. From germline transgenic DNA-NLS-injected founders, 47% transgenic F1s are obtained in wild-type crosses, as opposed to 6% from naked DNA-injected founders (P < 0.01). In both cases, the transgene is transmitted to the F2 generation. In addition, high-resolution FISH analysis of transgenic F1s reveals that the use of NLS increases the number of distinct transgene integration sites along chromatin fibres.