FLPe functions in zebrafish embryos

To assay the efficiency of the FLP/FRT site-specific recombination system in Danio rerio, a construct consisting of a muscle-specific promoter driving EGFP flanked by FRT sites was developed. FLPe capped RNA was microinjected into transgenic single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 h post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; P < 0.001) than would be expected from a cross in the absence of the recombinase (50%). Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR analysis and FLPe-mediated recombination was confirmed by DNA sequencing. Sperm derived from confirmed transgenic males in these experiments was used to fertilize wild-type eggs to determine whether germline excision of the transgene had occurred. Clutches sired by FLPe-microinjected males contained 0–4% fluorescent embryos. Transgenic males that were phenotypically wild-type produced no fluorescent progeny, demonstrating complete excision of the transgene from their germline. FLPe microinjected males that retained some fluorescent muscle expression produced a small proportion of fluorescent offspring, suggesting that in mosaic males not all germline cells had undergone FLPe-mediated transgene excision. Our results show that FLPe, which is derived from Saccharomyces cerevisiae, is an efficient recombinase in zebrafish maintained at 28.5°C.     

Analysis of chromosomal equipment in spermatozoa of a 46,XY/47,XY/+8 male by means of multicolour fluorescent in situ hybridization: confirmation of a mosaicism and evaluation of risk for offspring

Trisomy 8 is generally associated with chromosomal mosaicism and occurs de novo, with relatively well-defined clinical manifestations, ranging from minimal effects to severe malformations. Mosaicism is often difficult to ascertain and the confirmation of diagnosis requires several chromosomal investigations on a variety of tissues. We present a case of mosaic trisomy 8 fortuitously found in a healthy 30-year-old man during a cytogenetic investigation for several spontaneous abortions: 8% of the lymphocyte metaphases showed a 47,XY,+8 karyotype. Fluorescent in situ hybridization (FISH) with the probes pJM.128 and L1.84 was performed on decondensed interphase spermatozoa. Of the 25 000 analysed cells, 398 spermatozoa (1.59%) exhibited a hybridization pattern compatible with a chromosome 8 disomy; the frequency was higher than in either sperm control populations (0.17% and 0.21%). This result is in agreement with the existence of trisomy 8 mosaicism in the gonads and germ cells. FISH on decondensed interphase spermatozoa spreads is thus an easy non-invasive procedure that can be used to complement mosaicism diagnosis in tissue other than blood. Moreover, FISH provides interesting data for characterizing the risk for offspring. Received: 11 July 1996 / Revised: 26 August 1996     

Electrofusion of mouse embryos results in uniform tetraploidy and not tetraploid/diploid mosaicism.

Some previous attempts to produce tetraploids experimentally have resulted in a proportion of treated embryos becoming 2n/4n mosaics at a frequency which may be as high as 20%, when using cytochalasin B as a fusigenic stimulus and cytogenetic techniques to identify putative tetraploid embryos. To investigate the possible occurrence of 4n/2n mosaicism, tetraploid embryos were produced by electrofusion, a process which allows adjacent blastomeres at the 2-cell stage to fuse following exposure to electric field pulses. Embryos used for electrofusion were hemizygous for a transgene consisting of approximately 1000 copies of the mouse beta-globin gene. After in situ hybridization, one hybridization signal is expected per diploid genome. Tetraploid cells in 7.5-, 8.5-, 9.5- and 10.5-day-old conceptuses were distinguished from diploid cells by performing in situ hybridization on histological sections. The frequency of nuclei with two hybridization signals in the 'hemizygous' tetraploid embryos was compared to diploid embryos which were either hemizygous or homozygous for the beta-globin transgene. Comparison of the frequency of nuclei with two hybridization signals between tissues of 'hemizygous' tetraploid conceptuses and homozygous diploid conceptuses showed no significant difference, which implies that the tissues in the tetraploid conceptuses were uniformly tetraploid. No evidence was found to suggest that electrofusion results in 2n/4n mosaicism.     

Identifying and genotyping transgene integration loci

The random germline integration of genetically engineered transgenes has been a powerful technique to study the role of particular genes in variety of biological processes. Although the identification of the transgene insertion site is often not essential for functional analysis of the transgene, identifying the site can have practical benefit. Enabling one to distinguish between animals that are homozygous or hemizygous for the transgene locus could facilitate breeding strategies to produce animals with a large number of genetic markers. Furthermore, founder lines generated with the same transgene construct may exhibit different phenotypes and levels of transgene expression depending on the site of integration. The goal of this report was to develop a rapid protocol for the identification and verification of transgene insertion sites. To identify host genomic sequences at the coagulation Factor X transgene integration site, DNA from a tail snip of the transgenic mouse was digested with NcoI and circularized using T4 DNA ligase. Using appropriately positioned PCR primers annealing to a transgene fragment distal to a terminal transgene restriction site (NcoI), one could amplify a fragment containing the transgene terminal region and extending into the flanking genomic sequence at the insertion site. DNA sequence determination of the amplicon permitted identification of the insertion site using a BLASTN search. FISH analysis of a metaphase spread of primary fibroblasts derived from the transgenic mouse was consistent with the identification of insertion site near the end of mouse chromosome 14. Identification of transgene insertion sites will facilitate genotyping strategies useful for the construction of mice with multiple engineered genetic markers and to distinguish among different founder lines generated by the same transgene. Furthermore, identification of the insertion site is necessary to analyze unexpected phenotypes that might be caused by insertional inactivation of an endogenous gene.     

Effect of transgene concentration, flanking matrix attachment regions, and RecA-coating on the efficiency of mouse transgenesis mediated by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) of DNA-loaded sperm cells has been shown to be a valuable tool for the production of transgenic animals, especially when DNA constructs with submegabase magnitude are used. In order to optimize and to understand the mechanism of the ICSI-mediated transgenesis, we have evaluated the impact of transgene DNA concentration, transgene flanking with nuclear matrix attachment regions (MARs), and the use of recombinase A (RecA)-coated DNA on the efficiency of mouse transgenesis production by ICSI. Presented data include assays with three DNA constructs; an enhanced green fluorescent protein (EGFP) plasmid of 5.4 kb, this plasmid flanked with two MAR elements (2.3 Kb of the human beta-interferon domain boundaries), and a yeast artificial chromosome (YAC) construct of ~510 kb (the largest transgenic construct introduced by ICSI that we have seen reported). ICSI-mediated transgenesis was done in the B6D2 mouse strain using different concentrations for each construct. Analysis of generated data indicated that ICSI allows the use of higher DNA concentrations than the ones used for pronuclear microinjection, however, when a certain threshold is exceeded, embryo/fetal viability decrease dramatically. In addition, independently of the transgene concentration tested, transgene flanking with MAR sequences did not have a significant impact on the efficiency of this transgenesis method. Finally, we observed that although the overall efficiency of ICSI-mediated transgenesis with fresh spermatozoa and RecA-complexed DNA was similar to the one obtained with the common ICSI-mediated transgenesis approach with frozen-thawed spermatozoa and RecA free DNA, this method was not as efficient in maintaining a low frequency of founder animal mosaicism, suggesting that different mechanisms of transgene integration might result from each procedure.     

Sexing river buffalo (Bubalus bubalis L.), sheep (Ovis aries L.), goat (Capra hircus L.), and cattle spermatozoa by double color FISH using bovine (Bos taurus L.) X- and Y-painting probes

River buffalo, sheep, and goat spermatozoa were cross-hybridized using double color fluorescence in situ hybridization (FISH) with bovine Xcen- and Y-chromosome painting probes, prepared by DOP-PCR of laser-microdissected-catapulted chromosomes, to investigate the possibility of using bovine probes for sexing sperm of other members of the family Bovidae. Before sperm analysis, the probes were hybridized on metaphase chromosomes of each species, as control. Frozen-thawed spermatozoa of cattle, river buffalo, sheep, and goat were decondensed in suspension with 5 mM DTT. Sperm samples obtained from three individuals of each species were investigated, more than 1,000 spermatozoa were scored in each animal. FISH analysis of more than 12,000 sperm revealed high level of sperm with X- or Y-signals in all of the species investigated, indicating FISH efficiency over 99%. Significant interspecific differences were detected in the frequency of aberrant spermatozoa (aneuploid and diploid) between goat (0.393%) and sheep (0.033%) (P < 0.01), goat and cattle (0.096%) (P < 0.5), as well as between river buffalo (0.224%) and sheep (P < 0.5). There was no significant difference between river buffalo and cattle. The present study demonstrated that it is possible to use bovine X-Y painting probes for sexing and analyzing sperm of other species of the family, thus facilitating future studies on the incidence of chromosome abnormalities in sperm as well as on sex predetermination of embryos for the livestock industry. Mol. Reprod. Dev. 67: 108-115, 2004.     

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn’t fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.     

Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM ± 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.     

Verification of flow cytometorically-sorted X- and Y-bearing porcine spermatozoa and reanalysis of spermatozoa for DNA content using the fluorescence in situ hybridization (FISH) technique

Flow cytometric sperm sorting based on X and Y sperm DNA difference has been established as the only effective method for sexing the spermatozoa of mammals. The standard method for verifying the purity of sorted X and Y spermatozoa has been to reanalyze sorted sperm aliquots. We verified the purity of flow-sorted porcine X and Y spermatozoa and accuracy of DNA reanalysis by fluorescence in situ hybridization (FISH) using chromosome Y and 1 DNA probe. Eight ejaculates from 4 boars were sorted according to the Beltsville Sperm Sexing method. Porcine chromosome Y- and chromosome 1-specific DNA probes were used on sorted sperm populations in combination with FISH. Aliquots of the sorted sperm samples were reanalyzed for DNA content by flow cytometry. The purity of the sorted X-bearing spermatozoa was 87.4% for FISH and 87.0% for flow cytometric reanalysis; purity for the sorted Y-bearing spermatozoa was 85.9% for FISH and 84.8% for flow cytometric reanalysis. A total of 4,424 X sperm cells and 4,256 Y sperm cells was examined by FISH across the 8 ejaculates. For flow cytometry, 5,000 sorted X spermatozoa and 5,000 Y spermatozoa were reanalyzed for DNA content for each ejaculate. These results confirm the high purity of flow sorted porcine X and Y sperm cells and the validity of reanalysis of DNA in determining the proportions of X- and Y-sorted spermatozoa from viewing thousands of individual sperm chromosomes directly using FISH.     

Simple Method of Zygosity Identification in Transgenic Mice by Real-time Quantitative PCR

To determine zygosity in transgenic (Tg) mice, a new technology, real-time quantitative PCR, has recently been introduced in transgenic research to overcome several drawbacks (time-consuming, specialized techniques and/or ambiguity in the results) of previously established methods, for example, Southern blot hybridization, dot blot hybridization, fluorescence in situ hybridization (FISH), etc. However, the previous real-time quantitative PCR method still possesses several drawbacks, for example, it needs two sets of primers/probes and the complicated setting up of appropriate conditions, both of which are expensive and remain time-consuming. We therefore developed an improved real-time quantitative PCR system for determination of zygosity, which is easy, rapid and less expensive, because the technique needs only two experimental processes: estimation of DNA concentration and CYBR Green PCR. We found that homozygous, hemizygous and non-Tg animals could easily be distinguished among F1 littermates in crosses of hemizygous EGFP- and DsRed2-Tg mice. Our improved method will be applicable to any Tg mouse strains, when a primer set is matched to the corresponding transgene.     

Nuclear Localization Signals Enhance Germline Transmission of a Transgene in Zebrafish

We report that cytoplasmic injection into zebrafish eggs of 104 copies of plasmid DNA complexed to nuclear localization signal (NLS) peptides, as compared to 106 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-injec...     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan     

应用荧光原位杂交检测EB病毒BNLF-1基因在转基因小鼠子代染色体上的整合及其定位

应用荧光原位杂交技术研究了EB病毒潜伏膜蛋白基因(BNLF-1)在转基因小鼠子二代染色体上的整合及其定位。结果在两只子二代转基因小鼠中,分别观察80个和60个分裂相,出现杂交信号的核型分别为27和18个,检出率为33.8％和30％。转基因分别整合在14号染色体和10号染色体上。提示转基因BNLF-1已稳定整合到转基因小鼠的染色体上,并通过生殖细胞遗传给子代;推测转基因原代鼠的转基因整合可能是随机的多位点整合。     

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.     

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

Male meiotic segregation of gonosomes analysed by two colour FISH in human interphase spermatozoa

Human meiotic segregation of X and Y chromosomes was simultaneously analysed by dual fluorescence in situ hybridization (FISH) on 10638 interphase spermatozoa from the same donor. A modified method for sperm decondensation ensured access of both X and Y probes to the sperm chromatin and a 99% hybridization efficiency. Expected sex ratios were obtained (49.30% haploidy X and 49.22% haploidy Y). The frequencies of meiotic II non-disjunctions for X and Y chromosomes (0.05%) were similar to those observed in sperm karyotypes after heterospecific fertilization of hamster eggs. In contrast, the frequency of XY bearing cells was significantly higher (0.42%). However, XY cells detected by FISH could either be diploid somatic cells, diploid germinal cells or hyperhaploid XY spermatozoa, the latter resulting from meiotic I non-disjunctions.     

Production of transgenic pigs mediated by pseudotyped lentivirus and sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency.     

Syndrome de klinefelter et conseil génétique

Klinefelter’s syndrome is a common sex chromosomal aberration generally characterized by hypergonadotrophic hypogonadism and azoospermia. However, spermatogenesis impairment is variable and severe oligozoospermia can be found in some men, particularly those exhibiting a mosaic karyotype 47,XXY/ 46,XY. New reproductive technologies, such as intracytoplasmic sperm injection (ICSI), allow Klinefelter patients to have a progeny, even those who are azoospermic after testicular sperm recovery. The question therefore arises of whether or not there is a genetic risk for pregnancies from affected fathers. Sperm karyotyping, by in vitro penetration of zona-free hamster eggs or by fluorescence in-situ hybridization (FISH), is a method of choice for measuring aneuploidy rate in spermatozoa of patients carrying gonosomal abnormalities. A theoretical model would predict a high level of 24,XX and/or 24,XY disomic sperm cells in Klinefelter patients if 47,XXY spermatogonia were able to complete meiosis and achieve spermatogenesis. Interestingly, current observations show that the rate of abnormal spermatozoa in these patients is low, around 1–2%, which indicates that only 46,XY spermatogonia can produce mature sperm cells and that oligozoospermic Klinefelter patients probably carry a 47,XXY / 46,XY mosaicism, at least at the testicular level. However, this low but statistically significant level of disomic spermatozoa emphasizes the fact that their spermatogenesis occurs in a compromised environment which could increase the risk of meiotic errors. Therefore, the possible occurrence of autosomal aneuploidies in children born from Klinefelter fathers leads to the following recommendations: a) individual analysis by FISH of the sperm aneuploidy rate in each Klinefelter patient candidate for ICSI; b) proposal of fetal karyotyping after amniocentesis in pregnancies obtained by this technique.     

A Cre/loxP-deleter transgenic line in mouse strain 129S1/SvImJ

A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.