生殖细胞及性腺移植

生殖细胞和性腺移植研究近年来已取得了突破性进展。这两项技术对于农业、医学及动物繁殖学的研究具有深远的意义和很大的应用价值。本文从同源移植、异源移植、移植技术及其它移植相关问题等方面对生殖细胞和性腺移植进行了简要介绍，并阐述了近年来在这方面所取得的进展。     

生殖细胞及性腺移植

生殖细胞和性腺移植研究近年来已取得了突破性进展。这两项技术对于农业、医学及动物繁殖学的研究具有深远的意义和很大的应用价值。本文从同源移植、异源移植、移植技术及其它移植相关问题等方面对生殖细胞和性腺移植进行了简要介绍,并阐述了近年来在这方面所取得的进展。     

鱼类遗传育种中生物学方法的应用及研究进展

鱼类遗传育种是指利用生物学方法对鱼类进行遗传选择或改造,从而获得新型改良鱼类的过程;它可以通过人为选择优势遗传性状或者通过整合或改变已有的遗传性状而达到遗传改良的目的.鱼类遗传育种是一个从稳定品系中筛选或研制变异品系,再从变异品系中培育稳定品系的循环过程.鱼类良种的培育成功将在很大程度上带动良种的饲养、加工、销售、休闲等后续产业的发展,这在渔业经济发展中具有重要意义.本文系统地总结了包括传统选择育种、分子标记辅助育种、全基因组选择育种和单性控制育种在内的选择育种技术,综述了杂交育种(近缘和远缘杂交)、细胞核移植、生殖干细胞和生殖细胞移植、人工雌核发育和雄核发育技术、多倍体育种在内的性状整合育种技术,以及以转基因育种为代表的性状改造育种技术,乃至这些育种技术在鱼类育种中的应用情况,同时结合本实验室在鱼类远缘杂交、雌核发育和雄核发育等染色体倍性育种方面的研究成果,对国内外鱼类育种的研究现状以及存在的问题进行了系统的概述.     

生物工程在水产养殖上的应用

生物工程既是一门综合性的新技术,又是一个知识密集的新产业。有人预言,生物工程将成为未来工业结构调整和改革的重要因素之一。我国是一个十亿人口的大国,农业是国民经济的基础,因此必须加强生物工程在农业应用方面的研究,包括基因重组、细胞融合、固定化酶和固定化细胞等技术,这将大大推动我国整个国民经济的发展。近年来,生物工程在水产养殖上的应用日益受到重视,并已见初效。现就鱼类性别的控制、缩短紫菜的生活史,细胞核移植和外源信使RNA注射、细胞融合、珍珠品质控制的可能性、鲍和鲑鳟的遗传工程,分述如下:     

美洲鲥雄性生殖细胞冷冻保存及移植

采用分离细胞冻存和组织块直接冻存2种方法, 进行美洲鲥(American shad, Alosa sapidissima)精巢细胞的长时间冷冻保存(>250d), 并比较分析2种不同冻存方法对美洲鲥雄性生殖细胞的冻存效果。解冻复苏后用Hochest33342和PI共染细胞核, 分析统计各期雄性生殖细胞的存活率, 结果显示组织块冻存方法所得精原干细胞和精母细胞的存活率明显高于分离细胞冻存的; 而精细胞及其他细胞存活率在2种方法间无显著差异; 特别是, 镜检发现组织块冻存方法所得精子存活率高达93.83%, 说明此冻存方法能同时高效地冻存美洲鲥各期生殖细胞, 包括成熟的精子。同时, 将组织块冻存的美洲鲥生殖细胞用PKH26染色标记后移植到出苗第1天的斑马鱼仔鱼中, 在细胞植入后5d仍能在受体中检测到供体细胞, 且有部分供体细胞能与内源生殖细胞共定位, 表明经过长时间冷冻保存的美洲鲥生殖细胞仍具有生殖细胞特性, 且能整合到斑马鱼受体性腺原基。研究结果为进一步开展美洲鲥, 或其他洄游性鱼类的生殖细胞发育、培养及种质资源保存等研究工作奠定了技术理论基础。     

鱼类生殖细胞

鱼类和其他大多数动物一样,胚胎发育产生两大细胞系,一个是生殖细胞系或种质系,另一个是体细胞系.生殖细胞系和体细胞系的分离发生在胚胎发育的早期,其标志是形成生殖细胞系的祖细胞,即原始生殖细胞(primordial germ cells,PGCs).PGC形成后从其"出生地"进行"长途跋涉"迁移到性原基,成为性原细胞,即卵原细胞和精原细胞;随后在经过配子生成的一系列发育过程后,最终产生成熟的配子——卵子和精子.生殖细胞发育的每个步骤都可能都可能影响生物个体的生殖能力或育性.巨大的生物学意义及其令人振奋的研究进展,使"生殖细胞"日益成为科学研究的热点,且作为特别议题出现在Science杂志2007年4月20日第5823期的封面上.近10年见证了对生殖细胞的认知的长足进步,这些都得益于对青鳉和斑马鱼等模式生物的研究.生殖细胞已能被准确地标记且分离以进行体外培养及移植,这为濒临绝种的动物借助近缘物种进行生殖繁衍提供了技术基础,譬如最近就有人成功地"借鲑鱼之腹"获得了虹鳟后代.另外,单倍体细胞体外培养已获成功,如最近有研究人员获得了青鳉鱼单倍体胚胎干细胞,而且把这种细胞的核移植到正常的卵子中,其能像正常精子授精一样发育并产生可育的子代.这种鱼其实最初是由嵌合卵发育而成,也就是由正常减数分裂生成的单倍体卵母细胞核和植入的体外培养的有丝分裂单倍体细胞核组成的卵.这种繁殖技术也被称作半克隆技术.这条首次获得的半克隆青鳉鱼被命名为"霍莉"(Holly).总之,本文将从基础研究和繁殖技术两方面,就生物界对鱼类生殖细胞研究和操作现状及未来研究方向进行小结与探讨.     

硬骨鱼类细胞核移植的研究进展

自１９９７年世界首例体细胞核移植绵羊“多莉”诞生以来,动物细胞核移植引起世界范围内人们的关注。硬骨鱼类具有很多优点,是研究核移植良好的材料。作者综述了硬骨鱼类细胞核移植技术的发展和完善以及在核质互作、细胞核发育全能性、鱼类育种及纯系建立等方面的应用,并对鱼类核移植存在的问题和前景进行了概括。     

多倍体鲫鲤原始生殖细胞的标记及其迁移研究

原始生殖细胞(primordial germ cells, PGCs)的起源和迁移已在多种鱼类进行了研究,但多倍体鲫鲤原始生殖细胞的标记和迁移尚未见报道。本实验室前期通过远缘杂交方法成功获得了一个倍性明确、亲缘关系清晰的多倍体杂交鱼研究体系,该体系由异源四倍体鲫鲤及其二倍体父母本和三倍体杂交后代组成。本文利用RNA定点表达(localized RNA expression, LRE)技术,将来自于斑马鱼的nanos1-3'-UTR与GFP融合后生成的m RNA注射入多倍体鱼受精卵中,首次对多倍体鲫鲤原始生殖细胞进行标记,并观察了其迁移途径。结果显示,多倍体鲫鲤PGCs能被斑马鱼GFP-nanos1-3'-UTR标记,并且多倍体鲫鲤PGCs的迁移与斑马鱼类似。本实验结果为多倍体鲫鲤原始生殖细胞的产生和迁移的研究提供了一些基础数据。     

鱼类染色体转移—仙台病毒融合胚胎细胞核移植的初步研究

细胞杂交技术近二十年来取得了巨大的成 就,现已被广泛地应用于医学和生物学各个领 域[4,51。但是体细胞杂交只能得到杂交的细胞, 还不能得到杂交的个体,至少在动物界是如此。 在植物方面已经能通过原生质体的融合产生杂 交细胞,再通过杂交细胞的培养产生新的再生 植株〔2,71。而动物的繁殖只能由生殖细胞通过 胚胎发育才能产生新的个体。虽然不少人曾做 了许多尝试,想通过卵子和体细胞人工融合的 方法产生杂种,但迄今还没有成功［[8-10,1310童第 周等11,11,121在鱼类细胞核移植方面做了不少工 作,利用鱼类囊胚细胞的核,移植到不同属和不 同亚科的去核未受精成熟卵内,得到了几种核 质杂种鱼。这些工作不但证明了鱼类早期囊胚 细胞具有发育的全能性,同时也说明利用动物 早期胚胎的体细胞核育种的可能性。那么用囊 胚细胞的融合引进外来的染色体以及改造原有 细胞的遗传组分,然后把融合的囊胚细胞的核 移植到去核的未受精的成熟卵里去,是不是能 产生具有新的遗传性状的个体呢？本实验正是 基于这样的目的进行的。首先我们使用同源的 囊胚细胞融合,然后移植核,以了解：(1)鱼类 囊胚细胞融合的条件;(2)融合后的囊胚细胞核 移植是否能促使卵子发育;(3）发育的胚胎染色 体组成如何？以便为导人异源染色体创造条件。     

果树种质资源超低温保存研究进展

综述了国内外在超低温保存果树种质资源方面的最新研究成果,从材料选择、材料预处理、冰冻保护剂、冰冻方法及超低温保存对组织超微结构的影响等几个方面总结了果树超低温保存的原理、影响因素及关键操作技术,展望了果树种质资源超低温保存技术的应用前景.     

Derivation and applications of human hepatocyte-like cells

Human hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) promise a valuable source of cells with human genetic background, physiologically relevant liver functions, and unlimited supply. With over 10 years’ efforts in this field, great achievements have been made. HLCs have been successfully derived and applied in disease modeling, toxicity testing and drug discovery. Large cohorts of induced pluripotent stem cells-derived HLCs have been recently applied in studying population genetics and functional outputs of common genetic variants in vitro. This has offered a new paradigm for genome-wide association studies and possibly in vitro pharmacogenomics in the nearly future. However, HLCs have not yet been successfully applied in bioartificial liver devices and have only displayed limited success in cell transplantation. HLCs still have an immature hepatocyte phenotype and exist as a population with great heterogeneity, and HLCs derived from different hPSC lines display variable differentiation efficiency. Therefore, continuous improvement to the quality of HLCs, deeper investigation of relevant biological processes, and proper adaptation of recent advances in cell culture platforms, genome editing technology, and bioengineering systems are required before HLCs can fulfill the needs in basic and translational research. In this review, we summarize the discoveries, achievements, and challenges in the derivation and applications of HLCs.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences that failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis. © 1996 Wiley-Liss, Inc.     

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.     

Future prospects for tissue engineered lung transplantation

The shortage of donor lungs for transplantation causes a significant number of patient deaths. The availability of laboratory engineered, functional organs would be a major advance in meeting the demand for organs for transplantation. The accumulation of information on biological scaffolds and an increased understanding of stem/progenitor cell behavior has led to the idea of generating transplantable organs by decellularizing an organ and recellularizing using appropriate cells. Recellularized solid organs can perform organ-specific functions for short periods of time, which indicates the potential for the clinical use of engineered solid organs in the future.

The present review provides an overview of progress and recent knowledge about decellularization and recellularization-based approaches for generating tissue engineered lungs. Methods to improve decellularization, maturation of recellularized lung, candidate species for transplantation and future prospects of lung bioengineering are also discussed.     

Future prospects for tissue engineered lung transplantation: Decellularization and recellularization-based whole lung regeneration

The shortage of donor lungs for transplantation causes a significant number of patient deaths. The availability of laboratory engineered, functional organs would be a major advance in meeting the demand for organs for transplantation. The accumulation of information on biological scaffolds and an increased understanding of stem/progenitor cell behavior has led to the idea of generating transplantable organs by decellularizing an organ and recellularizing using appropriate cells. Recellularized solid organs can perform organ-specific functions for short periods of time, which indicates the potential for the clinical use of engineered solid organs in the future.   The present review provides an overview of progress and recent knowledge about decellularization and recellularization-based approaches for generating tissue engineered lungs. Methods to improve decellularization, maturation of recellularized lung, candidate species for transplantation and future prospects of lung bioengineering are also discussed.     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.     

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.     

Animal board invited review: Germplasm technologies for use with poultry

Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted.