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 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. © 1995 wiley-Liss, Inc.     

Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.     

Use of coisogenic host blastocysts for efficient establishment of germline chimeras with C57BL/6J ES cell lines.

Gene targeting in embryonic stem (ES) cells allows the production of mice with specified genetic mutations. Currently, germline-competent ES cell lines are available from only a limited number of mouse strains, and inappropriate ES cell/host blastocyst combinations often restrict the efficient production of gene-targeted mice. Here, we describe the derivation of C57BL/6J (B6) ES lines and compare the effectiveness of two host blastocyst donors, FVB/NJ (FVB) and the coisogenic strain C57BL/6-Tyr(c)-2J (c2J), for the production of germline chimeras. We found that when B6 ES cells were injected into c2J host blastocysts, a high rate of coat-color chimerism was detected, and germline transmission could be obtained with few blastocyst injections. In all but one case, highly chimeric mice transmitted to 100% of their offspring. The injection of B6 ES cells into FVB blastocysts produced some chimeric mice. However; the proportion of coat-color chimerism was low, with many more blastocyst injections required to generate chimeras capable of germline transmission. Our data support the use of the coisogenic albino host strain, c2J, for the generation of germline-competent chimeric mice when using B6 ES cells.     

Genetic analyses of plumage color mutations on the Z chromosome of Japanese quail

Genetic analyses were performed with four sex-linked plumage color mutations (roux, brown, imperfect albino, and cinnamon) in Japanese quail (Coturnix japonica). Roux and brown quail have similar plumage color, but plumage of roux quail is paler. Pure, F1 and F2 matings were carried out with roux and brown stocks, and 357, 338, and 273 progeny with either roux or brown plumage color were obtained from each mating type, respectively. These allelism tests showed that mutations for roux and brown colors were alleles (*R and *B) from the same locus BR, and that BR*B was dominant over BR*R. Two alleles at the AL locus, AL*A (imperfect albino) and AL*C (cinnamon) were used to estimate the recombination frequency between the BR and AL loci on the Z chromosome. It was estimated to be 38.1+/-1.0% based on 4615 chicks from the test crosses.     

Functional gametes derived from explants of single blastomeres containing the "germ plasm" in Xenopus laevis: a genetic marker study

Single blastomeres containing the "germ plasm" were isolated from 32-cell embryos of Xenopus albino (ap/ap) or wild type and cultured in vitro until the corresponding normal control embryos reached the neurula stage. The resulting explants from albinos were implanted into wild-type host neurulae and vice versa. The formation of functional gametes, eggs or sperm, of donor type was tested when the operated host embryos had reached sexual maturity. The color of the eggs laid by the experimental females and the presence or absence of melanophores in the epidermis and of pigment granules in the eyes of hatched larvae from matings of the experimental males with albino females made possible the identification of donor-type gametes. Twelve males and 12 females of the wild-type hosts, and 16 males and 14 females of the albino hosts survived. Six animals produced donor-type eggs or sperm, most of them being germ line chimeras. This shows that functional gametes can develop from explants derived from single blastomeres containing the "germ plasm."     

Development and Initial Characterization of Xenomitochondrial Mice

Xenomitochondrial mice harboring trans-species mitochondria on a Mus musculus domesticus (MD) nuclear background were produced. We created xenomitochondrial ES cell cybrids by fusing Mus spretus (MS), Mus caroli (MC), Mus dunni (Mdu), or Mus pahari (MP) mitochondrial donor cytoplasts and rhodamine 6-G treated CC9.3.1 or PC4 ES cells. The selected donor backgrounds reflected increasing evolutionary divergence from MD mice and the resultant mitochondrial-nuclear mismatch targeted a graded respiratory chain defect. Homoplasmic (MS, MC, Mdu, and MP) and heteroplasmic (MC) cell lines were injected into MD ova, and liveborn chimeric mice were obtained (MS/MD 18 of 87, MC/MD 6 of 46, Mdu/MD 31 of 140, and MP/MD l of 9 founder chimeras, respectively). Seven MS/MD, 1 MC/MD, and 11 Mdu/MD chimeric founder females were mated with wild-type MD males, and 18 of 19 (95%) were fertile. Of fertile females, only one chimeric MS/MD (1% coat color chimerism) and four chimeric Mdu/MD females (80-90% coat color chimerism) produced homoplasmic offspring with low efficiency (7 of 135; 5%). Four male and three female offspring were homoplasmic for the introduced mitochondrial backgrounds. Three male and one female offspring proved viable. Generation of mouse lines using additional female ES cell lineages is underway. We hypothesize that these mice, when crossbred with neurodegenerative-disease mouse models, will show accelerated age-related neuronal loss, because of their suboptimal capacity for oxidative phosphorylation and putatively increased oxidative stress.     

Exclusive transmission of the embryonic stem cell‐derived genome through the mouse germline

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Production of quail (Coturnix japonica) germline chimeras derived from in vitro-cultured gonadal primordial germ cells

A previous report from our laboratory documented successful production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells (gPGCs). Subsequently, this study was designed to evaluate whether gPGCs can be maintained in vitro for extended period, and furthermore, these cultured PGCs can induce germline transmission after transfer into recipient embryos. In experiment 1, gonadal cells from the two strains (wild-type plumage (WP) and black (D) quail) were cultured in vitro for 10 days. Using antibody QCR1, we detected a continuous, significant (P = 0.0002) increase in the number of WP, but not D, PGCs. QCR1-positive WP colonies began to form after 7 days in culture. On Day 10 of culture, 803 WP PGCs were present as a result of a continuous increase, whereas no D PGC colonies could be detected and the D gonadal stroma cells were rolled up. Differences in the PGCs or the gonadal stroma cells of the two different strains might account for these differences. In experiment 2, WP PGC colonies were maintained in vitro up to Day 20 of culture, and 10- or 20-day-cultured PGCs were microinjected into dorsal aortas of 181 recipient D embryos. Thirty-five (19.3%) of the transplanted embryos hatched after incubation, and 25 (71.4%) of the hatchlings reached sexual maturity. Testcrossing of the sexually mature hatchlings resulted in three (10 days, 33.3%) and eight (20 days, 50.0%) germline chimeras respectively. This report is the first to describe successful production of germline chimera by transfer of in vitro-cultured gPGCs in quail.     

The production of chimeric rats and their use in the analysis of the hooded pigmentation pattern

New, improved media and procedures for making rat chimeric embryos and culturing them in vitro have been developed. We have produced 27 rat chimeras: 20 males and 7 females. This ratio of males to females is consistent with that seen in mouse chimeras, suggesting that rat sex chimeras develop as phenotypic males. By aggregating embryos containing appropriate genetic markers for pigment cell differentiation, it is possible to produce chimeras that elucidate the site of action of the hooded gene. The coat color patterns of black ? black hooded chimeras display a white belly spot. In black ? albino hooded chimeras, small patches of white hair appear on the head and a large white spot occurs on the belly. Black ? agouti hooded chimeras display both agouti and nonagouti pigmentation over the entire surface of the chimera. These animals are fully pigmented with no white spots. In black ? albino non-hooded chimeras, rather small irregular patches of black and white hairs are distributed throughout the pelage. Histological examination of sections of hair follicles obtained from the white areas in the head of black ? albino hooded chimeras revealed amelanotic melanocytes. On the other hand, hair bulbs from the white belly spots do not contain any such melanocytes. Thus the white hairs of the head are due to the presence of albino melanocytes, but the white hairs of the belly are due to the total absence of melanocytes. All these observations are consistent with the conclusion that the hooded gene acts within melanoblasts, probably to retard their migration from the neural crest and/or to prevent their entrance into the hair follicles of the white areas of hooded rats.     

Strain preference in donor and recipient for production of W-bearing sperm in mixed-sex germline chimeric chickens

To elucidate the strain preference in donor and recipient for the production of W-bearing sperm, mixed-sex germline chimeric chickens were produced. The combination of donor and recipient was White Leghorn (WL) and Barred Plymouth Rock (BPR), and vice versa. Four sets of mixed-sex chimeras that had the male phenotype at sexual maturity were subjected to analysis: group 1, a female WL donor and a male BPR recipient; group 2, a male WL donor and a female BPR recipient; group 3, a female BPR donor and a male WL recipient; group 4, a male BPR donor and a female WL recipient. The mean number of W-bearing sperm detected by in situ hybridization among 10000 sperm observed was 135, 158, 26 and 71 in groups 1, 2, 3 and 4, respectively. The number in group 1 was significantly higher than that of group 3 (P<0.05). And the number in group 2 was significantly higher than those of groups 3 and 4 (P<0.05). It is suggested that the combination of a WL donor and a BPR recipient produced W-bearing sperm more efficiently than the reverse combination.     

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.     

Transmission of gametes with normal or translocation chromosomes in male and female single-sex mouse chimeras.

Three male and four female mouse single-sex chimeras derived from fusions of Rb(11.13)4Bnr T(1;13)70H homozygous embryos with +/+ embryos were caged with T(1;13)70H homozygotes of the opposite sex and followed through their reproductive lifespans. Six animals (three males and three females) were germline chimeras. The fz gene was used as a marker for the T70H reciprocal translocation. The ratio of fz/fz to fz/+ offspring did not change with increasing age in males, but decreased in two of the three female chimeras. Within males, there was generally good agreement between the proportions of translocation and nontranslocation germ cells from spermatogonial mitosis through the first and second meiotic division. In one male, this ratio was also reflected in the offspring. In the other two males, there was significant selection during haplophase, from which both types of spermatozoa could benefit.     

The Perfect Host: A Mouse Host Embryo Facilitating More Efficient Germ Line Transmission of Genetically Modified Embryonic Stem Cells

There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs). We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH) that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium). ESC germline transmission was observed in 9/11 (82%) lines using PH blastocysts, compared to 6/11 (55%) when conventional host blastocysts were used. Furthermore, less than 35% (83/240) of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137) of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the production of genetically modified animals.     

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus)

In our previous studies, we demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, to investigate the differentiation pattern of female PGCs in male gonads in chickens, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W-bearing spermatogonia, spermatocytes, and round spermatids accounted for 30.8%, 32.7%, and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second meiotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis.     

Plumage phenotypes and mate preferences in Japanese quail 2. sexual imprinting

Mate selection, with emphasis on early social (sexual imprinting) and subsequent long-term social experience, was studied in a randombred population of Japanese quail consisting of wildtype (W), redhead (R) and Albino (A) plumage colors. Early social experiences involved situations where flocks of the various plumage colors were maintained either separately or intermingled.Initial mate preferences were determined from a series of paired choice-tests between plumage phenotypes. Wildtype and redhead females exhibited no preferences, while albino hens preferred albino males. Preferences exhibited by albino males depended on sexual imprinting; those with no other experience preferred albinos and redheads to wildtypes, whereas those raised with other morphs did not distinguish among phenotypes. Redhead and wildtype males while avoiding albino hens, did not distinguish between redhead and wildtype hens.Combinations of the plumage color-social experience flocks (A&R; A&W; R&W) were housed for long-term observations of mate selection. Albino hens mated only albino males. Redhead and wildtype hens having previous experience with albinos mated more frequently with albino males than those lacking such experience. Redhead and wildtype hens showed no preference between redhead and wildtype males. Albino males did not distinguish among female plumage colors, whereas redhead and wildtype males avoided albino hens, and mated equally with redhead and wildtype hens. In a series of nonsimultaneous choice trials, redhead and wildtype females were mated significantly more than albinos. These results demonstrate the influence of genetic mechanisms, sexual imprinting and subsequent long-term social experiences on the optimization of mate selection.     

Genetic control of pest Lepidoptera: construction of a balanced lethal strain in Ephestia kuehniella

A genetic method for the suppression of Lepidopteran pests has been investigated in the Mediterranean flour moth, Ephestia kuehniella Zeller. The method is based on the release of males trans-heterozygous for two sex-linked recessive lethal mutations (SLRLMs). In this paper, characteristics of 16 new SLRLMs are presented. The construction of a balanced lethal strain, BL-2, which was the last step to develop the method, is reported. Males of the strain are balanced for two non-allelic SLRLMs, sl-2 and sl-15. Females carry either sl-2 or sl-15 in their Z chromosome and the T(W;Z)2 translocation on their W chromosome. The translocation includes wild-type alleles of both lethal loci so that the females are viable. Matings between males of the BL-2 strain and normal females of the wild-type strain gave 99.74% male progeny. Exceptional females were due to recombination between the sl-2 and sl-15 loci. Thus, males of the BL-2 strain have a potential to suppress wild populations of the pest. Another envisaged use of this method is for an effective sexing technique.     

Comparison of male chimeric mice generated from microinjection of JM8.N4 embryonic stem cells into C57BL/6J and C57BL/6NTac blastocysts

To identify ways to improve the efficiency of generating chimeric mice via microinjection of blastocysts with ES cells, we compared production and performance of ES-cell derived chimeric mice using blastocysts from two closely related and commonly used sub-strains of C57BL/6. Chimeras were produced by injection of the same JM8.N4 (C57BL/6NTac) derived ES cell line into blastocysts of mixed sex from either C57BL/6J (B6J) or C57BL/6NTac (B6NTac) mice. Similar efficiency of production and sex-conversion of chimeric animals was observed with each strain of blastocyst. However, B6J chimeric males had fewer developmental abnormalities involving urogenital and reproductive tissues (1/12, 8?%) compared with B6NTac chimeric males (7/9, 78?%). The low sample size did not permit determination of statistical significance for many parameters. However, in each category analyzed the B6J-derived chimeric males performed as well, or better, than their B6NTac counterparts. Twelve of 14 (86?%) B6J male chimeras were fertile compared with 6 of 11 (55?%) B6NTac male chimeras. Ten of 12 (83?%) B6J chimeric males sired more than 1 litter compared with only 3 of 6 (50?%) B6NTac chimeras. B6J male chimeras produced more litters per productive mating (3.42?±?1.73, n?=?12) compared to B6NTac chimeras (2.17?±?1.33, n?=?6). Finally, a greater ratio of germline transmitting chimeric males was obtained using B6J blastocysts (9/14; 64?%) compared with chimeras produced using B6NTac blastocysts (4/11; 36?%). Use of B6J host blastocysts for microinjection of ES cells may offer improvements over blastocysts from B6NTac and possibly other sub-strains of C57BL/6 mice.     

Sex determination in the androdioecious plant Datisca glomerata and its dioecious sister species D. cannabina.

Datisca glomerata is an androdioecious plant species containing male and hermaphroditic individuals. Molecular markers and crossing data suggest that, in both D. glomerata and its dioecious sister species D. cannabina, sex is determined by a single nuclear locus, at which maleness is dominant. Supporting this conclusion, an amplified fragment length polymorphism (AFLP) is heterozygous in males and homozygous recessive in hermaphrodites in three populations of the androdioecious species. Additionally, hermaphrodite x male crosses produced 1:1 sex ratios, while hermaphrodite x hermaphrodite crosses produced almost entirely hermaphroditic offspring. No perfectly sex-linked marker was found in the dioecious species, but all markers associated with sex mapped to a single linkage group and were heterozygous in the male parent. There was no sex-ratio heterogeneity among crosses within D. cannabina collections, but males from one collection produced highly biased sex ratios (94% females), suggesting that there may be sex-linked meiotic drive or a cytoplasmic sex-ratio factor. Interspecific crosses produced only male and female offspring, but no hermaphrodites, suggesting that hermaphroditism is recessive to femaleness. This comparative approach suggests that the hermaphrodite form arose in a dioecious population from a recessive mutation that allowed females to produce pollen.     

Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number

 Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis. This is reflected by the fact that the ES cell technology to date could only be fully developed in mice. In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism. Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation. The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts. Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution. During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells. Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells. Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka. Received: 5 June 1998 / Accepted: 6 July 1998