Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos.

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Livestock embryo sexing: A review of current methods, with emphasis on Y-specific DNA probes.

Control of the sex ratio of domestic species is potentially of great commercial importance to agriculture. While sexing of spermatozoa would be the most advantageous approach, studies to date suggest that this technology is unlikely to be available in the near future. As an alternative, four methods of sexing embryos have been developed. The use of X-linked enzymes and a serological assay involving H-Y antigen are noninvasive methods which have the advantage of allowing all embryos to be sexed, but these methods are not always accurate. Cytogenetic analysis and the use of Y-specific DNA probes are invasive methods which are limited by the accessibility of embryonic material for biopsy, but they are highly accurate. Each method is reviewed, with an emphasis on the use of Y-linked probes, and each is seen to have both advantages and limitations; the difficulty is in achieving a method that provides both an accurate sexing procedure and an acceptable pregnancy rate after embryo transfer. While no single method currently available fulfills all the criteria for a commercial method of embryo sexing, the potential for the development of an ideal method does exist.

Normal levels of vitamin A in tissues of the Syrian hamster (Mesocricetus auratus) determined colorimetrically.

Vitamin A levels in tissues of 20 normal adult hamsters on a standard diet were measured colorimetrically. No significant difference between male and female animals was found for any of the tissues sampled. The mean vitamin A value for blood plasma in 20 animals was 53.4 micrograms/dl. Mean values for liver, kidneys, flank skin and cheek pouch were 813, 1.29, 1.84 and 1.31 mg/g wet weight, respectively. The vitamin assay was less suitable for small organs such as trachea.

Increased clearance rate of plasma thyroxine accompanies estradiol-induced vitellogenesis in male Japanese quail.

Four days after giving 16 mumol estradiol-17 beta (E2) per 100 g body weight to male Japanese quail, the thyroids of these quail accumulated twice as much 125I as did the thyroids of control quail. Plasma thyroxine concentrations were 7.38 +/- 45 (SE) ng/ml (n = 16) for the E2-treated quail and 3.38 +/- .53 ng/ml (n = 16) for the controls. Clearance rates of plasma thyroxine were 534 ng thyroxine/100 g/day for the E2-treated quail and 234 ng/100 g/day for the control quail. The E2 may have increased the metabolism of thyroxine in these male quail and, by inference, stimulated a twofold increase in the rate of synthesis and secretion of thyroxine.

Demethylation of the gene expressing a yolk protein precursor in quail.

Japanese quail produce more egg-yolk protein per gram of body weight than chickens do. Because cytosine phosphoguanine (CpG) methylation has been reported to correlate inversely with gene activity, the demethylation of cytosine residues in the quail vitellogenin 2 (Vg2) gene was assessed as a possible contributing factor. Vitellogenin genes are normally transcribed in the liver of female Japanese quail that are sexually mature. Changes in the methylation pattern of CCGG sites of the quail Vg2 gene were studied using the methyl-sensitive restriction endonucleases Msp I and Hpa II. At least five CCGG sites were detected within the transcribed region of the Vg2 gene; one or two of these sites were hypomethylated in all tissues examined in male and female quail, two sites underwent transcription-dependent demethylation in the liver of laying females and also in the liver of males following estradiol injection (64 mumol estradiol/mL of absolute ethanol at .25 mL/100 g of BW), and another site was demethylated in the liver of estradiol-injected males but not in laying females. Two further sites present within the 5' flanking sequences or 5' structural region underwent transcription-dependent demethylation in the liver of laying females and of estradiol-treated males; one or both of these sites appears to be demethylated in the oviduct. Another site, within the 3' flanking sequences or the 3' structural gene region, underwent estradiol-dependent demethylation in the liver and the oviduct. The expression-linked demethylation of the CCGG sites in the Vg2 gene of quail appears to be similar to that reported for the chicken VTGII gene. The results of this study did not explain the enhanced production of yolk protein in Japanese quail, but did suggest the presence of a fundamental, conserved mechanism that must play an important role in the expression of the avian Vg gene.

An immunological study of the diverse forms of vitellogenin identified in quail plasma.

Quail vitellogenins (which exist in three different native forms designated Vg alpha, Vg beta and Vg gamma) can be separated into two fractions by DEAE-cellulose chromatography: peak III (containing Vg alpha, Vg beta, and Vg gamma) and peak IV (containing Vg alpha and Vg beta). In the present study, antibodies were prepared against peak III and peak IV vitellogenin fractions, which were then compared with each other and with vitellogenin-containing plasma by immunodiffusion, rocket immunoelectrophoresis, and two-dimensional immunoelectrophoresis. Peak III and peak IV vitellogenin fractions behaved similarly on immunodiffusion and gave an apparently homogeneous precipitin band. However, rocket immunoelectrophoresis revealed at least three or four antigenic determinants in peak III or peak IV vitellogenin fractions. Two-dimensional immunoelectrophoresis showed that peak III vitellogenin fractions contained an antigenic determinant not present in peak IV vitellogenin. In addition, some constituents within any vitellogenin-containing sample had antigenic determinants in common, as revealed by the continuity of certain precipitin peaks, indicating a possible structural relatedness between Vg alpha, beta, and/or gamma. Plasma from untreated male quail had an antigenic determinant in common with all vitellogenin-containing plasma samples.

A comparison of diethylstilbestrol- and estradiol-17 beta-induced vitellogenesis in quail.

Japanese quail were injected intramuscularly with .125 to 32 mumole/100 g body weight of estradiol-17 beta (E2) or diethylstilbestrol (DES). During the following 8 days, vitellogenesis was assessed in these birds by measuring plasma concentrations of protein-bound phosphorus (PBP; vitellogenin is a phosphoprotein), calcium, and total protein. Blood packed cell volumes were recorded also. Plasma PBP concentrations of E2-injected male and female quail increased to .83 and .96 mg/ml from control values of .004 and .12 mg/ml, respectively. Doses of 8 to 32 (males) or 4 to 32 (females) mumole E2/100 g body weight induced similar initial rates of vitellogenesis, but the duration of vitellogenesis increased with increasing doses of hormone. In male quail, low doses of DES (.5 to 4.0 mumole/100 g body weight) were more effective than equal doses of E2 in inducing increases in plasma PBP levels. However, the maximum PBP concentrations attained with high doses of DES were less than those attained with equal doses of E2 (.53 and .83 mg/ml, respectively).

Induction of vitellogenesis in Japanese quail as a sensitive indicator of the estrogen-mimetic effect of a variety of environmental contaminants.

Vitellogenesis was induced in mature male Japanese quail following intramuscular injections of 16 mumol/100 g body weight of any one of four estrogen analogues or 160 mumol/100 g of the nonsteroid zearalenone. Six hours after the injections, microgram levels of vitellogenin were detected and quantitated by rocket immunoelectrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of plasma from estrogen-treated birds and from 160 mumol zearalenone-treated birds showed, 4 days after the injections, the three subunits of vitellogenin normally seen in the plasma of egg-laying female quail. As evidenced by increased concentrations of plasma protein-bound phosphorus, total plasma calcium, and total plasma protein, mole-for-mole diethylstilbestrol dipropionate was 114%, ethynyl estradiol 75%, moxestrol 108%, and zearalenone 5.6% as effective as estradiol-17 beta in inducing vitellogenesis over a 4-day period. The responses to 160 mumol zearalenone/100 g over 8 days were approximately .6 the responses for estradiol. The responses to ethynyl estradiol were about equal to those for estradiol for days 1 and 2, then faded to .3 the estradiol response by day 8. Doses of 16 or 160 mumol/100 g of cholecalciferol, chlordecone, corticosterone, o,p1-DDT [1,1,1-trichlor-2-(p-chlorphenyl)-2-(o-chlorphenyl)eth ane], methoxychlor, progesterone, or testosterone, or 16 mumol/100 g of zearalenone did not induce vitellogenesis. Induction of vitellogenesis in male Japanese quail can thus provide a sensitive test of the estrogen-mimicking activity of some possible environmental contaminants.

153Gadolinium as a useful radiolanthanide for long-term labeling of tissues in Japanese quail.

153Gadolinium (153Gd), with gamma energies of 97 keV (30%) and 103 keV (20%), and a nuclear-life of 242 days and with no primary charged particles, was selected as the radiolanthanide for labeling proteins and tissues in Japanese quail. Ranges of dose-responses 18 hr after giving Gd (153Gd labeled) were: males, livers 24.4% for .00015 mumol/100 g to 63.8% for .15 mumol/100 g; laying females, livers 6.5% for .00015 mumol/100 g to 72.7% for 15 mumol/100 g and growing oocytes 83.2% for .00015 mumol/100 g to 6.4% for 15 mumol/100 g. Accumulation of .015 mumol/100 g doses of 153Gd in tissues common to both sexes were greater in males than in females at 1, 6, and 18 hr. The exception was the pancreas, where for all three sampling times the percentages for females were twice those for males. Comparison of 153Gd with 140lanthanum (140La) showed 18-hr levels of 27%/g liver in males and 1.6%/g liver and 11.8%/g growing oocyte in females for both nuclides. Double-labeling with 153Gd and 144cerium (144Ce) showed that a near-toxic dose of La (15 mumol/100 g) caused a marked decrease in labeling of growing oocytes (e.g., largest oocyte, La-treated females, Gd 14.6%, Ce 6.1%; controls Gd 42.2%, Ce 35.5%) and a marked increase in labeling of livers (La-treated, Gd 51.2%, Ce 43.3%; controls; Gd 7.62%, Ce 4.75%).

Manipulation of blastodermal cells.

Blastodermal cells isolated from newly laid, unincubated eggs are virtually uncommitted cells that exhibit many of the properties of pluripotential stem cells. They can be transferred from donor to recipient embryos and contribute to both somatic tissues and the germline. Blastodermal cells that have been maintained in culture for 7 d express the epitopes ECMA-7 and SSEA-1, which are also expressed by mouse embryonic stem cells. After culture for up to at least 7 d, blastodermal cells retain the ability to differentiate into somatic tissues and the germline both in vivo and in vitro. Proliferation in the absence of differentiation of blastodermal cells is stimulated by the presence of Leukemia Inhibitory Factor (LIF) and other ligands that interact with the gp130 receptor, and differentiation is stimulated by exposure to retinoic acid. Blastodermal cells also possess high levels of telomerase activity, which is shared by immortalized cells and cells within the germline. Blastodermal cells can be transfected and will express foreign genes both in vivo and in vitro. Transfected cells can be isolated by fluorescence activated cell sorting and can be cryopreserved without losing their ability to contribute to either somatic tissues or the germline. These properties of blastodermal cells make them ideal vectors for introducing genetic modifications to the germline.

Chimeric chickens and their use in manipulation of the chicken genome.

Germline chimeric chickens can be made by injecting dispersed cells from Stage X blastoderms into recipient embryos at an equivalent stage of development. Colonization of the chimera by donor-derived cells is facilitated when the recipient embryo is compromised by exposure to irradiation prior to injection of the donor cells. Donor cells can be genetically manipulated by lipofection-mediated gene transfer before they are introduced into the recipient. The genetic modification is expressed in the ectoderm, mesoderm, and endoderm of the chimera after incubation for 96 h. Donor cells can also be cultured as dispersed cells in a monolayer or as whole-embryo explants for at least 48 h before transfer into recipients and retain the ability to enter both somatic and germline tissues in the resulting chimera. A strategy is proposed for the production of transgenic chickens using lipofection-mediated gene transfer to blastoderm cells isolated from Stage X embryos, which are subsequently injected into compromised recipients to yield a germline chimera.

Lack of excision of introns from primary transcripts of certain chicken vitellogenin II minigenes.

Two truncated versions of the chicken vitellogenin II gene VTGII were designed and constructed to include all known essential regulatory elements of the complete gene. Both pCB123 and pCB123/4 contain 945 base pairs (bp) of the 5'-flanking sequence, introns and exons 1-3, and a subset of the remaining 32 exons of VTGII, inserted into a pBluescript SK (+/-) vector. pCB123/4 contains 752 bp of legitimate VTGII 3'-flanking sequences, while the 3' end of pCB123 terminates at the VTGII cDNA end, followed by AT-tailing and vector sequences carried over during cloning. Expression of these plasmids was tested following their lipofection into primary cultures of chicken hepatocytes established from day 14 embryos. Poly(A)+ RNA derived from pCB123 was detected by Northern blotting and reverse transcription-polyacrylamide chain reaction. No evidence was observed for appropriate hormonal control of expression, despite the presence of 17 beta-estradiol or colipofection with the estrogen receptor clone pHEO. VTGII sequences at the 3' end of pCB123/4 led to an apparent destabilization of the RNA transcript. Unexpectedly, unprocessed pCB123 transcripts of varying lengths accumulated in the cells. These experiments constitute the first reported attempts to express authentic VTGII coding sequences in cultured cells and highlight the dilemma of which introns to include in a minigene. Despite reports that some minigenes are expressed more efficiently if one or two introns are included, other minigenes may be expressed more effectively in the absence of introns. In the case of a complex gene with many introns, such as VTGII, there may be a preferential order in which introns are removed from the primary construct. The truncation of complex genes to give functional minigenes for transgenic studies may require considerable experimentation.

Apparent absence of oct 3/4 from the chicken genome.

The development of efficient methods for establishing germline competent chicken embryonic stem (ES) cell lines has proved elusive. In the mouse embryo, expression of oct 3/4 is limited to pluripotent cells and primordial germ cells; regulatory sequences of this gene have been used to derive germline competent mouse ES cell lines by the continuous ablation of differentiated cells in culture using drug selection. To apply this technique to chickens several strategies were employed to analyze the chicken genome for oct 3/4, a member of the highly conserved POU gene family. PCR and Southern hybridization experiments with primers and probes based on mouse oct 3/4 sequences indicated that oct 3/4-like sequences are not present in the chicken genome. Also, analysis of mRNA from Stage 14 and 20 (H&H) chick embryos by reverse transcription PCR and the screening of a Stage 20 (H&H) chick embryo cDNA library with mouse oct 3/4-based primers and probes indicated that oct 3/4-like sequences are not expressed in the early chick embryo. The apparent absence of oct 3/4 in chickens, despite the conservation of the gene in mammals and urodeles, is discussed in terms of possible implications for the mode of chicken PGC formation in relation to that in other vertebrates.

The chicken stathmin gene and its expression in the embryo.

Stathmin, which functions as an intracellular relay in signal transduction pathways, has been suggested as a potential indicator of pluripotent cells in the early mouse embryo. In this study, chicken stathmin cDNA and genomic DNA were analyzed. In mammals stathmin consists of five exons and four introns; exons 3, 4, and 5 in the mammalian stathmin gene are equivalent to one relatively large exon in the chicken stathmin gene. Introns equivalent to introns 3 and 4 in the mammalian stathmin gene are not present in the counterpart gene in chickens and, although intron 2 was shown to be present in both mammals and birds, it is smaller in the chicken stathmin gene. Despite differences in the genomic organization of the gene and its smaller size in chickens compared with that in humans and mice, similarities in the coding sequences and in the expression of the chicken and mouse stathmin genes at certain stages of embryo development, as determined by whole-mount in situ hybridization experiments, suggest that their products are functional homologues. The argument is thus substantiated for further investigations into the use of regulatory regions of the stathmin gene in a system for the establishment of long-term cultures of germline competent chicken embryonic stem (ES) cells by the selective ablation of differentiated cells in culture using drug selection.

Construction and characterization of a large-fragment chicken bacterial artificial chromosome library.

A large-insert bacterial artificial chromosome (BAC) library has been constructed from male chicken genomic DNA using the new pBeloBAC11 vector. The library was prepared in two parts, such that two-thirds of the BAC library (2976 clones) had an average insert size of 490 kb (80 clones analyzed), after optimization of transformation and HMW DNA size-selection conditions. Fragments of increased average size were cloned by coupling a second size strategy using pulsed-field gel electrophoresis with optimized electroporation that favored transformation of Escherichia coli DH10B cells with very large plasmids. The initial one-third of this library (1440 clones) was constructed using the standard protocols and had an average insert size of 180 kb (40 clones analyzed). The overall library consists, at present, of 4416 clones with a combined insert size average of 390 kb (ranging from 25 to 725 kb). At least 95% of the BAC clones contain inserts. This is partially due to a second color selection performed with respect to white colonies, as well as to the optimized ligation conditions used. Based on the percentage of clones with inserts and the analysis of insert sizes, we estimate this library to represent a 0.8-fold coverage of the chicken genome. Southern blot analysis and fluorescence in situ hybridization were performed to confirm the identity of the BAC inserts with chicken genomic DNA. Analysis of large chicken BAC inserts showed that they were stably propagated for at least 120 cell generations. The results indicate that the BAC system is able to carry stably very large genomic fragments of chicken DNA, this system translating into a powerful tool for physical mapping and positional cloning of the chicken genome.

Relatedness among Rhizobium and Agrobacterium species determined by three methods of nucleic acid hybridization.

Deoxyribonucleic acid (DNA) was isolated from 20 strains of Rhizobium and Agrobacterium and from one strain of Serratia marcescens; the guanine plus cytosine content of each DNA sample was determined by thermal denaturation. Radioactive DNA was isolated from three reference strains following the uptake of [2-(14)C]thymidine in the presence of deoxyadenosine. Ribonucleic acid (RNA) polymerase was used to synthesize radioactive RNA on DNA templates from the three reference strains. Radioactive DNA and RNA from the three reference strains were each hybridized with filter-bound DNA from all of the 21 test strains in 6 x SSC (standard saline citrate) and 50% formamide at 43 C for 40 hr. DNA/DNA relatedness was also determined by spectrophotometric measurement of the rates of association of single-stranded DNA. The order of relatedness between strains was similar by each method. Overall standard deviations for the DNA/DNA and DNA/RNA membrane filter techniques were +/-0.87 and +/-1.03%, respectively; that for the spectrophotometric technique was +/-4.11%. The DNA/DNA membrane technique gave higher absolute values of hybridization than did the DNA/RNA technique. R. leguminosarum and R. trifolii could not be distinguished from each other by these techniques. These results also indicated close relationships between R. lupini and R. japonicum, and (with less certainty) between R. meliloti and R. phaseoli. Of all the rhizobia tested against the A. tumefaciens 371 reference strain, the R. japonicum strains were the most unrelated. The three Agrobacterium strains used were as related to the R. lupini and R. leguminosarum references as were several rhizobium strains.

Comparative study of the physiology of vitellogenesis in Japanese quail.

1. During a study of vitellogenesis in the Japanese quail, male or female quail were given, intramuscularly, one to three doses of estradiol-17 beta (16 mumol/100 g body weight). 2. In treated males, the plasma concentrations of protein-bound P (PBP, a measure of the phosphate groups in vitellogenin), Ca, and total protein fluctuated in unison. 3. Packed cell volume (PCV) values varied approx, inversely as the PBP concentrations. 4. Maxima of approx. 0.85 mg PBP/ml occurred six, five, and four days after the primary, secondary, and tertiary injections. Following each maximum, the PBP concentrations declined rapidly to the 0.004 mg/ml level found in the plasma of untreated male quail. 5. During periods of elevated PBP levels, Ca and PBP were present in equimolar quantities. 6. When laying female quail were given a single injection of estradiol, the PBP concentration rose from 0.12 to 0.97 mg/ml. 7. PCV values for both male and female estrogen-treated quail decreased from approx. 0.50 to 0.30. The decreases in PCV values were attributed to increases in plasma volume as measured using 125I-labelled serum albumin. No marked change in total erythrocyte mass was measured using 51CrO2-(4) as the cell label. 8. Perhaps the decrease in plasma serum albumin concentrations reported by some investigators for estrogen-treated animals is due to dilution rather than to repression of serum albumin synthesis.

Persistence of added retinoids in organ culture media during induction of mucous metaplasia and glandular morphogenesis in hamster cheek pouches.

The retinoid concentration (determined colorimetrically) did not change significantly in retinyl acetate-supplemented (6 micrograms/ml) Eagle's Minimal Essential Medium containing 10% fetal calf serum when stored at -20 or 4 degrees C over 7 days. After the medium was incubated at 37 degrees C for 48 h, 37-49% of the retinoid remained, whether or not tissue (neonatal Syrian hamster cheek pouch) was present, and irrespective of explant age. The normal retinoid level in the tissue was approximately 0.25 micrograms per gram. Therefore, neonatal hamster cheek pouches, incubated in medium with the addition of 6 micrograms of retinyl acetate per ml of medium and undergoing mucous metaplasia and some mucous gland morphogenesis, were continually being exposed to retinoid levels which, though gradually decreasing, remained well above their normal physiological level.