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1.
鸡X期胚盘细胞体外培养   总被引:4,自引:0,他引:4  
杜立新  尹春光 《动物学报》2002,48(4):549-553
为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后,直接显微注入同期受体胚盘(140枚);或对转染后供体细胞进行G418抗性筛选性后显微注入同期受体鸡胚盘(140枚);或将供体细胞体外培养48h,再与脂质体-P^GSI复合物共孵育后显微注入同期受体鸡胚盘(190枚),制备转基因嵌合体鸡,并应用PCR和RAPD方法,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明:直接注射组孵化率(5.7%)显著(P<0.01)高于G418筛选处理组(1.4%)和培养48h处理组(2.1%);G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明,体外培养48h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力,利用早期胚盘细胞途径制备转基因鸡是可行的。  相似文献   

2.
转ω-3脂肪酸去饱和酶基因猪在优质猪培育及研究ω-3不饱和脂肪酸预防心血管和癌症疾病中的作用方面有着重大的应用.本研究首次通过体细胞核移植制备了转线虫ω-3脂肪酸去饱和酶基因sFat-1猪.将sFat—1基因转染到大白猪胎儿成纤维细胞,获得转基因阳性细胞克隆,然后以转基因细胞为核供体、体外成熟的猪卵母细胞为核受体构建克隆胚胎,胚胎体外培养或进行移植.先后移植了1889枚1-4细胞期克隆胚胎到10头受体母猪的输卵管内,28天B超检测9头受体母猪妊娠(90%),7头妊娠足月(70%)分娩产下21头仔猪,体细胞克隆猪的效率为1.1%(出生仔猪/移植胚胎).体细胞克隆猪效率的提高,主要是对克隆胚胎的移植环节进行了改进,比较了受体母猪排卵状况对胚胎移植效率的影响.当受体母猪卵泡发育处于即将排卵或正在排卵阶段,能够获得较高的妊娠率和妊娠足月率(100%),而排卵后移植妊娠足月率为0%.对健康存活15头克隆猪进行了PCR和Southern检测,证实13头为转基因猪,转基因阳性率为87%.RT—PCR检测13头转基因猪,12头表达sFat—1基因.以上结果表明,利用优化的体细胞转基因结合核移植技术,可以成功地批量生产转sFat-1基因的克隆猪.  相似文献   

3.
鸡Ⅹ期胚盘细胞体外培养   总被引:9,自引:0,他引:9  
杜立新  尹春光 《动物学报》2002,48(4):549-553
为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力 ,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后 ,直接显微注入同期受体胚盘 (14 0枚 ) ;或对转染后供体细胞进行G418抗性筛选后显微注入同期受体鸡胚盘 (14 0枚 ) ;或将供体细胞体外培养 4 8h ,再与脂质体 PGS1复合物共孵育后显微注入同期受体鸡胚盘 (190枚 ) ,制备转基因嵌合体鸡 ,并应用PCR和RAPD方法 ,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明 :直接注射组孵化率(5 7% )显著 (P <0 0 1)高于G418筛选处理组 (1 4 % )和培养 4 8h处理组 (2 1% ) ;G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明 ,体外培养 4 8h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力 ,利用早期胚盘细胞途径制备转基因鸡是可行的。  相似文献   

4.
小鼠ES细胞种系嵌合体的获得   总被引:14,自引:0,他引:14  
陈伟胜  韩嵘 《遗传学报》1999,26(2):126-134
种系嵌合体的获得是实现ES细胞介导的转基因途径的决定步骤,ES细胞种系分化能力的保持是决定种系嵌合的前提条件,而事体的主种系嵌合体的获得则是判定ES细胞系是否具有种系分化能力的唯一方法,为考察本室新近建立的3种小鼠ES细胞系MESPU21.MESPU22和MESPU29的种系分化能力,选用近交系C57BL/6J及远交系KMW和ICR为受体胚胎提供者,分别通过囊胚注射法和8细胞期桑椹胚注射法进行了嵌  相似文献   

5.
兔体细胞核移植的初步研究   总被引:4,自引:0,他引:4  
实验以兔胎儿成纤维细胞为核供体,对兔体细胞核移植技术的融合,激活和发育等环节进行了初步研究。实验通过比较不同电场强度对兔2细胞胚胎卵裂球融合以及卵母细胞激活的影响,证实200和260V/mm的电场强度可有效地诱导2细胞胚胎的融合和兔卵母细胞的孤雌激活。然后将200和260V/mm电场强度用于体细胞核移植,融合率分别为44.4%和48.4%,卵裂率分别为58.8%和53.8%,桑椹胚/囊胚发育率分别为5.9%和5.5%。但112枚核移植胚胎移植到5只受体后没有幼子出生。结果表明,实验中所建立的程序至少可以支持兔体细胞克隆胚胎的早期发育。  相似文献   

6.
家兔供体细胞的发育周期与重构胚发育的关系   总被引:3,自引:0,他引:3  
采用血清饥饿法处理体外培养的兔子胎儿成纤维细胞,并将其作为供体细胞移入去核卵母细胞内构建重构胚胎。检查供体细胞的细胞周期对重构胚的融合率、分裂率和着床率的影响。实验结果表明:培养基中血清含量在0.5%的情况下,G0/G1期的细胞比例由正常培养条件下(培乔液中含有10%FCS)的73.2%明显地增加到86%以上。饥饿1~3天的细胞作为供体细胞构建重构胚时,可明显提高重构胚的融合率,但是不同的饥饿时间其融合率并无显著的差异。饥饿处理可明显增加重构胚的分裂率,以饥饿处理3天为最佳。  相似文献   

7.
为了提高异种间核移植重构胚的发育率,本研究以体内排放的奶山羊成熟卵为供胞质的受体细胞,以人、兔、波尔山羊等的异种或亚种体细胞的原代核移植(Primary Somatic Cell Nuclear Transfer,PSCNT)重构早胚(8-16细胞期)的卵裂球作供核体,观察经亚种或异种卵胞质体短期“修饰”的核再移植产生的继代(Secondary SCNT,SSCNT)重构胚的着床前发育潜能。结果:人、兔、波尔山羊的继代桑椹/囊胚发育率均显著地高于其PSCNT胚胎(人,14.81%VS.7.79%;兔,23.53%VS.12.50%;波尔羊,55.35%VS.24.53%);这些早胚的各阶段发育时程仍遵循供核体动物正常受精卵的发育时程。结果启示:奶山羊成熟卵胞质对异种体细胞核亦具一定的去分化能力,能支持重构胚发育到囊胚;异种重构胚的发育特征是由供体核所决定的;继代核移植几乎能够成倍提高异种间重构胚的着床前发育率,提示核的去分化完全是在母型信息主导的调控之下完成的,而进一步发育的时序似乎是由核决定的:成倍延长在含母型信息主导调控环境中的时间能成倍提高SCNT重构胚的着床前发育率。  相似文献   

8.
对进口X祖代鸡B系和D系共143羽异性公鸡的K座位羽速型作了K座位隐性测交,杂合子占41.96%,慢羽纯合子占35.66%,另有9羽和2羽公鸡分别因后裔数少于7羽或是后裔快慢羽鸡比例严重偏离1:1而被判为无效,对8羽进口祖代D系异性公鸡的PCR-RFLP分析的结果表明,6羽公鸡为K座位显性纯合子(仅检到1450bpDNA片段),2羽为杂合子(检到1450bp、1068bp和382bp共3条带)。通  相似文献   

9.
具高效种系嵌合能力的C57BL/6J 小鼠ES细胞系的建立   总被引:10,自引:2,他引:8  
童英  韩嵘  郑玉兰  苏平  尚克刚 《遗传学报》1999,26(5):468-473
采用小鼠原代成纤维细胞作为饲养层,在含1×103单位白血病抑制因子(LIF)的DMEM高糖培养基中,建成了11个C57BL/6J小鼠的ES细胞系,成系率为9.6%。所建的11个ES细胞系中有5个核型正常率大于70%。这些细胞具早期胚胎细胞的特征,呈碱性磷酸酶阳性,具表达0014基因的特性5进行体内分化实验时能发生广泛的分化。通过嵌合体制作实验证明其中3个系具嵌合能力,并从中筛选出具高效种系嵌合能力的MESPU35细胞系,MESPU35细胞的克隆能达到种系传递,经过基因操作的细胞克隆,也保留了高效的嵌合能力。因此,MESPU35细胞可作为制作突变小鼠的有效载体。  相似文献   

10.
慢性前列腺炎患者假丝酵母菌感染及耐药性分析   总被引:1,自引:1,他引:0  
目的 了解慢性前列腺炎与假丝酵母菌感染的相关性及假丝酵母菌的耐药性。方法 采用常规沙堡平板分离360例慢性前列腺炎患者的前列腺液标本中的假丝酵母菌,疑似菌落用ATB Expression鉴定仪进行鉴定。采用ATB-Fungus真菌药敏板,对假丝酵母菌株进行药敏试验。结果 11.7%前列腺液标本(42/360)似丝酵母菌阳性,其中自假丝酵母菌23例(54.7%),近平滑假丝酵母菌13例(30.9%),其它6例(14.3%)。假丝酵母菌菌株对两性霉素B和制霉菌素敏感率均为100%,其次为酮康唑,敏感率为97.0%;对5-氟胞嘧啶耐药性最强,其耐药率为56.5%。结论 白假丝酵母菌和近平滑假丝酵母菌是慢性前列腺炎假丝酵母菌感染的优势菌种,假丝酵母菌株最敏感的药物是两性霉素B和制霉菌素。  相似文献   

11.
In the avian species, germline chimera production could be possible by transfer of donor germ cells into the blood vessel of recipient embryos. This study was conducted to establish an efficient transfer system of chicken gonadal primordial germ cells (gPGCs) for producing the chimeras having a high capacity of germline transmission. Gonadal PGCs retrieved from 5.5-day-old embryos (stage 28) of Korean Ogol chicken (KOC with i/i gene) were transferred into the dorsal aorta of 2.5-day-old embryos (stage 17) of White Leghorn chicken (WL with I/I gene). Prospective evaluations of whether culture duration (0, 5, or 10 days) and subsequent Ficoll separation of gPGCs before transfer affected chimera production and germline transmission in the chimeras were made while retrospective analysis was conducted for examining the effect of chimera sexuality. A testcross analysis by artificial insemination of presumptive chimeras with adult KOC was performed for evaluating each treatment effect. First, comparison was made for evaluating whether experimental treatments could improve chimera production, but none of the treatments were significantly (P = 0.6831) influenced (5.1%-14.4%). Second, it was determined whether each treatment could enhance germline transmission in produced chimeras. More (P < 0.0001) progenies with black feathers (i/i) were produced in the germline chimeras derived from the transfer of 10-day-cultured gPGCs than from the transfer of 0- or 5-day-cultured gPGCs (0.6%-7.8% vs. 10.7%-49.7%). Ficoll separation was negatively affected (P < 0.0001), whereas there was no effect in chimera sexuality (P = 0.6011). In conclusion, improved germline transmission of more than a 45% transmission rate was found in chicken chimeras produced by transfer of 10-day-cultured gPGCs being separated without Ficoll treatment.  相似文献   

12.
This study reports for the first time the production of chicken germline chimeras by transfer of embryonic germ (EG) cells into recipient embryos of different strain. EG cells were established by the subculture of gonadal tissue cells retrieved from stage 28 White Leghorn (WL) embryos with I/I gene. During primary culture (P(0)), gonadal primordial germ cells (gPGCs) in the stromal cells began to form colonies after 7 days in culture with significant (P < 0.0001) increase in cell population. Colonized gPGCs were then subcultured with chicken embryonic fibroblast monolayer for EG cell preparation. Prepared EG cells or gPGCs at P(0) were transferred to stage 17 Korean Ogol chicken (KOC) embryos with i/i gene. The recipient chickens were raised for 6 months to sexual maturity, then a testcross analysis by artificial insemination was conducted for evaluating germline chimerism. As results, transfer of EG cells and gPGCs yielded total 17 germline chimeras; 2 out of 15 (13.3%) and 15 of 176 sexually matured chickens (8.5%), respectively. The efficiency of germline transmission in the chimeras was 1.5-14.6% in EG cells, while 1.3-27.6% in gPGCs. In conclusion, chicken germline chimeras could be produced by the transfer of EG cells, as well as gPGCs, which might enormously contribute to establishing various innovative technologies in the field of avian transgenic research for bioreactor production.  相似文献   

13.

Background

The Houbara bustard (Chlamydotis undulata) is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.

Methodology/Principal Findings

Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs) was injected into White Leghorn chicken (Gallus gallus domesticus) embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16) gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.

Conclusion

This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian species that cannot breed in captivity.  相似文献   

14.
We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.  相似文献   

15.
In the chicken, Dominant white is one of the major loci affecting feather color. Germline chimeric chickens are identified by testcross analysis using this genetic marker. The testcross, however, is a time-consuming and laborious procedure, resulting in the need for a faster and simpler molecular method. A recent study showed that Dominant white was exclusively associated with a 9-bp insertion in the PMEL17 gene. We searched for breed-specific sequence polymorphisms in the PMEL17 gene among White Leghorn (WL) (white feather), Korean Ogol Chicken (KOC) (black feather), and Barred Plymouth Rock (grayish-white, each feather regularly crossed with parallel blue-black bars). In addition to the 9-bp insertion, WLs and KOCs have unique bases in single nucleotide polymorphisms (SNPs) at the 1,777th and 3,118th bases in the PMEL17 gene. To detect these sequence polymorphisms, allele-specific polymerase chain reaction (AS-PCR) was performed, which successfully distinguished the different breeds. We confirmed the ability of the AS primers to detect germline chimerism. This simple method can be widely used for the screening of germline chimeric chickens.  相似文献   

16.
Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.  相似文献   

17.
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.  相似文献   

18.
Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.  相似文献   

19.
Feather melanocytes in the Barred Plymouth Rock (BPR) and White Leghorn (WL) chickens die prematurely in vivo when compared to the wild type Jungle Fowl (JF) chicken. Since these mutant melanocytes live in vitro, an environmental factor in the feather must precipitate their death. Results show that the addition of selected antioxidants, glutathione (GSH) and superoxide dismutase (SOD), can rescue these mutant melanocytes in vitro that have been placed under stress conditions that cause their premature cell death. Measurements of in vivo levels of GSH, catalase, and SOD show no significant difference in catalase activity between the JF, BPR, and WL feathers but do show a significant reduction in GSH activity in both the BPR and WL feathers to approximately 66% of the GSH concentration found in JF feathers. SOD activity in the BPR tissue is reduced significantly to approximately 50% of the JF activity and the WL SOD activity is reduced significantly to approximately 50% of the BPR SOD activity. Preliminary results of measurements of glutathione peroxidase activity indicate there is no difference in the levels of this enzyme in JF, BPR and WL feathers. A working hypothesis, based on current results, is proposed for premature cell death in BPR and WL feather melanocytes. The BPR melanocytes are genetically sensitive due to a defect in their SOD and GSH levels caused by the barring gene (B) and their death, due to reactive species of oxygen radicals, is precipitated in the poorly vascularized feather by the accumulation of oxygen radicals due to the low turnover of tissue fluids. The WL chicken carries the dominant white gene (I) in addition to the B gene. This gene directs the further reduction of the level of SOD and, when combined with the cell death mechanism already present in the BPR chicken, causes the WL feather melanocytes to die much earlier than the BPR feather melanocytes which in turn die much earlier than the wild type JF melanocytes. This same mechanistic hypothesis could apply as a cause of premature melanocyte cell death in human vitiligo wherein the vitiliginous melanocytes may have a genetic defect in their oxygen radical protection system.  相似文献   

20.
Ha JY  Park TS  Hong YH  Jeong DK  Kim JN  Kim KD  Lim JM 《Theriogenology》2002,58(8):1531-1539
We previously reported that germline chimeras could be produced by transfer of chicken gonadal primordial germ cells (gPGCs) cultured for a short term (5 days). This study was subsequently undertaken to examine whether gPGCs maintained in vitro for an extended period could retain their specific characteristics to induce germline transmission. Chicken (White Leghorn, WL) gPGCs were retrieved from embryos at stage 28 (5.5 days of incubation) and continuously cultured for 2 months in modified Dulbecco's minimal essential medium without subpassage and changing of the feeder cell layer. After the identification of gPGC characteristics using Periodic acid-Shiff's (PAS) reaction and anti stage-specific embryonic antigen-1 (SSEA-1) antibody staining at the end of the culture, cultured gPGCs were injected into the dorsal aorta of Korean Ogol Chicken (KOC) recipient embryos at stage 17 (2.5 days of incubation). Nineteen chickens (13 males and 6 females) were hatched, grown to sexual maturity, and subsequently subjected to testcross analysis employing artificial insemination with adult KOC. Of these, four (three males and one female) hatched chickens with white coat color. The percentage of germline chimerism was 21% (4/19). The results of this study demonstrated that gPGCs could maintain their specific characteristics for up to 2 months in vitro, resulting in the birth of germline chimeras following transfer to recipient embryos.  相似文献   

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