A high-resolution radiation hybrid map of porcine chromosome 6.

A high-resolution comprehensive map was constructed for porcine chromosome (SSC) 6, where quantitative trait loci (QTL) for reproduction and meat quality traits have been reported to exist. A radiation hybrid (RH) map containing 105 gene-based markers and 15 microsatellite markers was constructed for this chromosome using a 3000-rad porcine/hamster RH panel. In total, 40 genes from human chromosome (HSA) 1p36.3-p22, 29 from HSA16q12-q24, 17 from HSA18p11.3-q12 and 19 from HSA19q13.1-q13.4 were assigned to SSC6. All primers for these gene markers were designed based on porcine gene or EST sequences, and the orthologous status of the gene markers was confirmed by direct sequencing of PCR products amplified from separate Meishan and Large White genomic DNA pools. The RH map spans SSC6 and consists of six linkage groups created by using a LOD score threshold of 4. The boundaries of the conserved segments between SSC6 and HSA1, 16, 18 and 19 were defined more precisely than previously reported. This represents the most comprehensive RH map of SSC6 reported to date. Polymorphisms were detected for 38 of 105 gene-based markers placed on the RH map and these are being exploited in ongoing chromosome wide scans for QTL and eventual fine mapping of genes associated with prolificacy in a Meishan x Large White multigenerational commercial population.

Expression of luteinizing hormone genes in bovine conceptuses.

RT-PCR analysis demonstrated that bovine conceptuses at days 16, 23 and 30 expressed LH-beta-like and glycoprotein hormone alpha-like transcript sequences; adult kidney, liver and brain produced predominantly unspliced products. Sequencing of the LH-beta-like fragment (from conceptuses at day 30) indicated complete homology with the published sequence. In addition, ribonuclease protection assay of RNA samples from bovine conceptuses at day 30 with a bovine LH-beta probe revealed the presence of protected molecules that appeared to be full length. Northern blot analysis of total RNA from conceptuses at day 30 failed to demonstrate the presence of LH-beta or glycoprotein alpha subunit transcripts, whereas both transcripts were readily detected in adult pituitary RNA. Administration of hCG into the uterus of heifers from day 14 to day 16 of the oestrous cycle did not affect circulating progesterone concentrations, whereas the same dose increased progesterone concentrations (P < 0.05) when administered intravenously. These results indicate that the early bovine conceptus transcribes genes encoding LH-alpha and -beta subunits, but at a level unlikely to be of physiological consequence.

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.

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.

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.

Generation of chicken Z-chromosome painting probes by microdissection for screening large-insert genomic libraries.

A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10-15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200-800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.

Microisolation of the chicken Z chromosome and construction off microclone libraries.

A simple method was used to adapt a standard light microscope for the collection of chicken Z chromosomes from mitotic-metaphase spreads. The DNA of the collected chromosomes was enzymatically amplified using a partially degenerate primer. The resulting sequences, within a size range of 200-800 bp, were cloned to produce a Z chromosome DNA library, using blunt-end ligation into a SmaI-digested pUC18 plasmid (the SureClone system; Pharmacia, U.S.A.). The microcloning experiments produced 1250 clones; the size range of the cloned inserts was 250-800 bp, with an average of 480 bp (176 clones examined). Using male chicken genomic DNA as a probe, 10 out of 17 randomly selected clones showed strong positive signals on Southern blots, confirming the origin of the inserts as chicken DNA. In addition, the Z-chromosome origin of a selected microclone was verified in a semiquantitative Southern blot hybridization that showed positive signals with intensities that were approximately twice as strong for male (ZZ) as for female (ZW) chicken genomic DNA when the clone was used as a probe. The value of these libraries in further analysis of the chicken Z chromosome is discussed.

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture.

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a 60Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germline transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally.

Sexual differentiation of chimeric chickens containing ZZ and ZW cells in the germline.

The developmental fate of male and female cells in the ovary and testis was evaluated by injecting blastodermal cells from Stage X (Eyal-Gliadi and Kochav, 1976: Dev Biol 49:321-337) chicken embryos into recipients at the same stage of development to form same-sex and mixed-sex chimeras. The sex of the donor was determined by in situ hybridization of blastodermal cells to a probe derived from repetitive sequences in the W chromosome. The sex of the recipient was assigned after determination of the chromosomal composition of erythrocytes from chimeras at 10, 20, 40, and 100 days of age. If the sex chromosome complement of all of the erythrocytes was the same as that of blastodermal cells from the donor, the sex of the recipient was assumed to be the same as that of the donor. Conversely, if the sex-chromosome complement of a portion of the erythrocytes of the chimera differed from that of the donor blastodermal cells, the sex of the recipient was assumed to differ from that of the donor. Injection of male blastodermal cells into female recipients produced both male and female chimeras in equal proportions whereas injection of female cells into male recipients produced only by male chimeras. One phenotypically male chimera developed with a left ovotestis and a right testis although sexual differentiation was usually resolved into an unambiguous sexual phenotype during development when ZZ and ZW cells were present in a chimera. Donor cells contributed to the germline of 25-33% of same-sex chimeras whereas 67% of male chimeras produced by injecting male donor cells into female recipients incorporated donor cells into the germline. When ZW cells were incorporated into chimeric males, W-chromosome-specific, DNA sequences were occasionally present in DNA extracted from semen. To examine the potential of W-bearing spermatozoa to fertilize ova, males producing ZW-derived offspring and semen in which W-chromosome-specific DNA was detected by Southern analysis were mated to sex-linked albino hens. Since sex-linked albino female progeny were not obtained from this mating, it was concluded that the W-bearing sperm cells were unable to fertilize ova. The production of Z-derived, but not W-derived, offspring from ZW spermatogonia indicates that female primordial germ cells can become spermatogonia in the testes. In the testes, ZW spermatogonia enter meiosis I and produce functional ZZ spermatocytes. The ZZ spermatocytes complete the second meiotic division, continue to differentiate during spermiogenesis, and leave the seminiferous tubules as functional spermatozoa. By contrast, the WW spermatocytes do not appear to complete spermiogenesis and, therefore, spermatozoa bearing the W-chromosome are not produced. When cells from male embryos were incorporated into a female chimera, ZZ "oogonia" were included within the ovarian follicles and the chromosome complement of genetically male oogonia was processed normally during meiosis. Following ovulation, the male-derived ova were fertilized and produced normal offspring. This is the first reported evidence that genetically male avian germ cells can differentiate into functional ova and that genetically female germ cells can differentiate into functional sperm.

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.

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)

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.

Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos.

Stage-X blastoderms, within intact eggs from White Leghorn hens, were exposed to 500-700 rads of gamma radiation from a 60Co source prior to injection, into the subgerminal cavity, of approximately 100 or 200-400 dispersed cells from stage-X blastoderms isolated from eggs laid by Barred Plymouth Rock hens. Embryos developing past day 14 of incubation and hatched chicks were assessed for donor and recipient cell contribution to the melanocyte population through examination of black and yellow down pigmentation, respectively (Barred Plymouth Rocks have a recessive allele at the I locus while the White Leghorns have a dominant allele at the I locus). Of the 809 embryos injected with approximately 100 cells, 192 developed past day 14 and black pigmentation, indicating somatic chimerism, was observed on 118 of the 192 (58%) embryos and chicks. Of the 296 embryos injected with 200-400 donor cells, 86 developed past day 14 of incubation. Somatic chimerism was observed on 55 of the 86 (64%) embryos and chicks. To test for germline chimerism, birds surviving to maturity were mated to Barred Plymouth Rocks. Five somatically chimeric females were produced when approximately 100 cells were injected, and one was a germline chimera. Six somatic female chimeras were produced following the injection of 200-400 cells, three of which proved to be germline chimeras by the presence of Barred Rock chicks among their offspring. Two of the nine males produced by injecting approximately 100 cells were germline chimeras.(ABSTRACT TRUNCATED AT 250 WORDS)

The fate of female donor blastodermal cells in male chimeric chickens.

In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)

Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)