Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

Research on the appropriate way to transfer exogenous substances into chicken embryos

In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation. Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice. However, there are few studies about the effect of the different injection site and dosage on chicken embryos. The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model. Freshly laid eggs （Rugao yellow chicken） were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively. Egg hatching rate was recorded and compared among injection sites and different doses. A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed. The SPSS （statistical package for the social science） software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages. The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses （P〈0.05）. The hatchability of the blunt end injection group was significantly higher than that of the other two sites. The egg hatching rate decreased with increased saline doses. The egg hatching rate of the 100 pL saline injection group was higher than the 200 and 300 μL dosage groups. Ultimately, we suggest that the optimal chicken embryo injection process is during early development, at the blunt end site with a dose less than 100 μL to minimize damage to the egg.

Effects of "Products of Chicken Embryo" on Growth and Sexual Development in Rats

Products of Chicken Embryo (PCE) such as Ji-Pei-Jing is a kind of food for Chinese children prepard from chicken embryo. Female rats on 21 days were administered with aqueous solutions of Ji-Pei-Jing (1. 2 %, 3 %, 12%, and 48%, respectively) by gavage up to their onsets of puberty.The rats in the control group were treated with distilled water. However, Ji-Pei-Jing treatment exerted some effects on sexual mauration in the immature female rats. Essentially, the effects showed a dose-response tendency with an inverted 'U' shape.The age of vopnal opening for gnup treated with Ji-Pei-Jing was significantly earier than that to the control. Its uterus weight/b. w. ratio also significantly increased on day 30 and at the first estrus. There were significantly increases in the adrenal weight/b. w. raio of 30-day-old rats that were treated with 3%, 12%, and 48% Ji-Pei-Jing. The rats treated with 48% Ji-Pei-Jing had significantly lesser ovary weight.b.w. radio on day 30, too. The rats treated with Ji-Pei-Jing could normally ovulate at the first estrus, and no significant differences were observed during estrous cycles.The effects of PCE on serum levels of K, P, LH in 30-day-old nds and FSH in 28-day-old ras were elevated significantly by 3 % Ji-Pei-Jing treatment. It appeare that the effects of PCE result from interaction of contained complex physiologically active substances. Steriods, especially estradio-17β,possibly Play a key role, and polpeptide hormones may also exert important effects.

Adaptability of Fowlpox Virus in Chicken Embryo Passage Cells

[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.

Chicken Embryo Inhibition Test of Recombinant Freeze-drying Chicken Interferon against Newcastle Disease Virus( NDV) F_(48)E_(10)

The paper was to study the inhibitory effect of recombinant freeze-drying chicken interferon against Newcastle disease virus （NDV） F48E10. Nine-day-old chicken embryos were inoculated with recombinant freeze-drying chicken interferon via allaotoic sack, while 10-day-old chicken embryos were inoculated with NDV F48E10, and in vivo protective efficacy of interferon on chichen embryos was studied. The results showed that the recombinant freeze-drying chicken interferon at the dose of 1.28 mg/embryo reached the protection ratio of 90% on chicken embryo infected by F48E10.

Hematomas and Limb Skeletal Malformations in Chicken Embryos Following Exposure to 5-Fluoro-2'-Deoxyuridine

Vascular injury or interruption may play a role in vertebrate limb teratogenesis. Since 5fluoro- 2'- deoxyuridine (FdU) can cause vascular injury in the murine limb and skull prior to the appearance of skeletal malformations in these structures, we studied the effects of this chemical on skeletal development in the chick embryo and noted any vascular injury. The yolk sacs of day three ehick embryos (Hamburger and Hamilton states 17-19) were injected with solutions of vary concentrations of FdU in saline. The embryos developed until the 10th day of incubation when they were fixed for study. Uninjected, saline injected, and sham injected control embryo were similarly fixed. Upon gross inspection, frequent diffuse and saccular hernatomas, as well as fluid-filled blisters, were noted in the limbs of embryos treated with FdU. After the embryos were fixed and cleared, and the skeletons stained, significant skeletal malformations were observed in these limbs. Bony elements of both the upper and lower limbs were affected in at least some of the embryos. The combination of FdU-induced hematomas and blisters with associated skeletal malformations in the same regions of some embryos suggests a relationship between these phenomena.

The Quality Characteristics of Eggs: Impact on the Volume of Allantoin-Amniotic Fluid of Chicken Embryos

In this paper, the eggs＇ weight, quality characteristics （ albumen density, shell quality, yolk size）, and egg weight loss during incubation having impact on volume of allantoin-anmiotic fluid are considered. The major parameters that should be taken into account when creating specialized population of chickens for the production of DEC （ developing embryos of chickens） are determined.

Glycerol monolaurate improves intestinal morphology and antioxidant status by suppressing inflammatory responses and nuclear factor kappa-B signaling in lipopolysaccharide-exposed chicken embryos

Medium-chain fatty acids and their derivatives are natural ingredients that support immunological functions in animals.The effects of glycerol monolaurate(GML)on intestinal innate immunity and associated molecular mechanisms were investigated using a chicken embryo model.Sixty-four Arbor Acres broiler embryos were randomly allocated into four groups.On embryonic day 17.5,the broiler embryos were administered with 9 mg of GML,which was followed by a 12-h incubation period and a12-h challenge with 32μg of lipopolysaccharide(LPS).On embryonic day 18.5,the jejunum and ileum were harvested.Results indicated that GML reversed the LPS-induced decline in villus height and upregulated the expression of mucin 2(P<0.05).GML decreased LPS-induced malondialdehyde production and boosted antioxidant enzyme activity(P<0.05).GML alleviated LPS-stimulated intestinal secretion of interleukin(IL)-1β,IL-6,and tumor necrosis factor-a(TNF-a)(P<0.05).GML also normalized LPS-induced changes in the gene expression of Toll-like receptor 4,nuclear factor kappa-B p65(NF-κB p65),cyclooxygenase-2,NOD-like receptor protein 3,IL-18,zonula occludens 1,and occludin(P<0.05).GML enhanced as well the expression of AMP-activated protein kinase a1 and claudin 1(P<0.05).In conclusion,GML improved intestinal morphology and antioxidant status by alleviating inflammatory responses and modulating NF-κB signaling in LPS-challenged broiler embryos.

shRNA-triggered RNAi inhibits expression of NDV NP gene in chicken embryo fibroblast

RNA interference(RNAi)technology is a powerful tool for identifying gene functions.Chicken embryo fibroblast(CEF)is an ideal model for studying the interaction between avian viruses and their hosts.To establish a methodological platform for RNAi studies in CEF,three plasmid vectors expressing short hairpin RNAs(shRNAs)targeted against the Newcastle disease virus(NDV)NP gene were constructed.One of them,ndv1,was proven effective on blocking viral replication in CEF and chicken embryos.Four hours prior to infection with NDV,the CEF was transfected with the plasmids by Silent-fect.An unrelated shRNA sequence(HK)was used in mock transfection.The expression of a potent shRNA resulted in up to 2.3,21.1 and 9.8 fold decreases in NP gene expression at 3,6 and 9 h post infection in CEF,respectively.The ndv1 was able to completely inhibit the replication of the virus in CEF within 48 post infection.Furthermore,the pathological changes in CEF caused by NDV were delayed,and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndv1.When the complex of shRNASilent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 10^(5) or 10^(6) ELD50 NDV,NDV replication was decreased by 94.14% and 62.15% after 17 h,respectively.These findings suggest that the newly synthesized NP protein is critical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.

Identification of the Sex of Earlier Embryos from Generic Hybrids of Chicken-Quail by Wpkci

In this study, a protocol was developed to identify the sex of earlier embryos of chicken （♂）-quail （♀） hybrids and successfully tested the sex proportion of each period （66-120 h）. We acquired cross bred eggs by artificial insemination, hatched them in the same batch according to the standard hatching condition of chicken, and collected earlier living embryos at 66, 72, 78, 84, 90, 96, 102, 108, 114, and 120 h randomly. We adopted RT-PCR protocol and multiple PCR, made the known sex quail as the external control, employed fl-actin as the internal control, and used primers that were designed according to conservative area of gene Wpkci of quail to identify the sex of earlier hybrid embryos. The results indicated that the primer of Wpkci can be used to identify the sex of hybrid embryos accurately; there were more male than female in earlier embryos, the sex proportion of earlier embryos compared with academic numerical value was significantly different （P〈0.01）, and there was no difference between different periods （P〉0.05）. In the present study, we concluded that a simple, fast, credible and stable protocol to identify the sex of earlier hybrids embryos had been established by using primer of Wpkci; in earlier embryos, the death rate of female was higher than that of male and there was no fluctuant peak.

抗鸡传染性支气管炎病毒中药的筛选

【目的】对鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)进行抗病毒中药的筛选,并验证其治疗效果,为临床用药提供一定参考。【方法】采用鸡胚培养法,通过预防和治疗2个角度对24味单一中药、6种市售复方中药进行抗IBV的有效成分或方剂筛选试验,即在SPF鸡胚中先注射中药2 h后再接种病毒和先接种病毒2 h后再注射中药。使用SPSS 20.0软件对鸡胚重量进行方差分析,利用实时荧光定量PCR法辅助判定治疗效果。【结果】对鸡胚净重进行方差分析显示,桔梗组与阳性对照组差异显著(P<0.05),与阴性对照组差异不显著(P>0.05),表明预防和治疗均有效果。实时荧光定量PCR检测鸡胚尿囊液病毒滴度结果显示,桔梗、鱼腥草、宣肺败毒方、绞股蓝、蒲公英、甘草对IBV增殖均有抑制作用,其中桔梗抑制效果最佳。【结论】桔梗对IBV有显著的预防及抑制效果,结果为深入研究桔梗作用机理提供了依据。

黄芩苷体外抗IBV QX株的作用研究

为研究黄芩苷体外抗禽传染性支气管炎病毒(Avian infectious bronchitis virus,IBV)QX株的效果,试验对IBV进行复壮与扩繁,制备原代鸡胚肾(CEK)细胞,并测定IBV对CEK细胞的TCID50和黄芩苷对CEK细胞的最大无毒浓度(MNTC);在MNTC基础上采用CCK法测定不同浓度黄芩苷对IBV的有效抑制率,观察不同浓度黄芩苷对IBV导致的CEK细胞的细胞病变效应(CPE)的抑制情况,并采用qPCR法测定不同浓度黄芩苷对IBV N基因相对表达量的影响。结果表明:成功复壮并扩繁了IBV,其未被其他病毒污染,对CEK细胞的TCID50为1×10^(-4.75)/0.1 mL;黄芩苷对CEK细胞的MNTC为9.75 mg/L;该浓度黄芩苷对IBV的有效抑制率最高,为64.03%,能有效减轻IBV对CEK细胞的CPE;各浓度黄芩苷均可以极显著降低CEK细胞中IBV N基因相对表达量(P<0.01)。说明黄芩苷对CEK细胞的MNTC为9.75 mg/L,该浓度黄芩苷具有较好的体外抗IBV QX株的作用。

广东规模化鸡场死鸡胚中鸡毒支原体的分离鉴定、致病性及药物敏感性

旨在从广东某规模化鸡场死鸡胚进行鸡毒支原体(Mycoplasma gallisepticum,MG)的分离,并进行遗传进化分析、致病性及药物敏感性研究。本研究从广东某规模化鸡场的带菌死胚卵黄组织中分离禽支原体,通过菌落观察、血清学试验、16S rRNA支原体通用引物序列鉴定等方法,对分离的菌株进行种属鉴定,同时将分离的毒株对鸡胚和SPF鸡进行攻毒试验及抗菌药物敏感性试验。结果显示:显微镜下菌落呈典型的“荷包蛋”状。平板凝集试验显示其与MG阳性血清有凝集反应,与MS阳性血清不反应;16S rRNA序列测序发现各分离株与MG相似性达99.9%,因此确定分离株为MG。将各分离株感染7日龄SPF鸡胚,结果显示感染鸡胚大多于临近出壳时死亡;感染3周龄的SPF鸡,3周后解剖发现鸡气囊发生显著病理变化,说明其具有较强致病力;药物敏感性试验结果显示,5株分离株对盐酸沃尼妙林、多西环素、泰万菌素及泰妙菌素有较高敏感性,对替米考星、泰乐菌素、恩诺沙星、红霉素、吉他霉素、林可霉素呈现出不同程度耐药性。综上,从广东某规模化鸡场死鸡胚中成功分离到5株MG分离株,各分离株均可引起红细胞凝集,导致鸡胚死亡,对SPF鸡可引起典型的气囊炎症状,且对多种药物存在不同程度耐药性。本研究为临床MG的分离鉴定及用药选择提供了可参考的技术方法及指导,为MG攻毒模型建立提供了研究基础。

不同禽胚和接种途径分离GoAstV JL01株的效果研究

鹅星状病毒(Goose astrovirus,GoAstV)可导致1~15日龄雏鹅痛风甚至死亡。为探究有效分离GoAstV方法,将RT-PCR鉴定为GoAstV阳性的雏鹅肝脏、肾脏研磨液通过尿囊腔、绒毛尿囊膜及卵黄囊途径分别接种至SPF鸡胚、SPF鸭胚及非SPF鹅胚,通过多次传代观察胚体剖检变化、死亡情况、核酸、病毒载量检测等,综合判断GoAstV JL01株在禽胚上的最佳分离方式。结果表明,病料经尿囊腔途径接种非SPF鹅胚及SPF鸭胚,绒毛尿囊膜途径接种非SPF鹅胚、SPF鸡胚及SPF鸭胚,卵黄囊途径接种SPF鸭胚均无法使胚体发生显著的剖检变化及规律性死亡,RT-PCR及RT-qPCR检测表明,病毒无法在相应胚体、尿囊液及胚体其他组织中有效增殖,卵黄囊途径接种SPF鸡胚所传的10代胚体均发生发育迟缓、出血等特征性病变,除F_(1)代其余9代均死亡,卵黄囊和胚体中病毒载量为10^(4.5)~10^(6.0)copies·μL^(-1)。因此,卵黄囊途径接种8日龄SPF鸡胚为GoAstV JL01株最适分离方式。研究为GoAstV分离提供可借鉴路径。

PTEN基因在鸡肝脏中的发育性变化

为了研究抑癌基因PTEN在家禽肝脏发育过程中的表达变化,试验选取50枚海兰褐受精蛋,在孵化的第15,18,20天[E15、E18、E20(啄壳为准)]和出壳后第1,5,10,15天(D1、D5、D10、D15)各取6枚(只)鸡胚/雏鸡,用乙醚麻醉后颈椎脱臼致死,取肝脏并提取基因组,采用实时荧光定量PCR和免疫组织化学方法对PTEN基因在家禽肝脏发育过程中的表达变化进行研究。结果表明:PTEN基因相对表达量随着胚龄/日龄的增长而增加,但在啄壳当天,PTEN基因相对表达量会暂时下降。PTEN蛋白在出壳前期主要分布在部分肝血窦内皮细胞的细胞核和肝细胞的细胞核中,在细胞质中基本无表达;在出壳后,PTEN蛋白在肝细胞核和细胞浆内均有分布。E20,免疫阳性PTEN蛋白的表达量下降,和E15水平相当(P>0.05),在其他胚龄/日龄时均显著高于E15(P<0.05)。肝细胞核PTEN蛋白免疫阳性率在E18显著高于E15(P<0.05);在E20和D15,肝细胞核PTEN蛋白免疫阳性率急剧下降,均显著低于E18(P<0.05);在D5~D15,肝细胞核PTEN蛋白免疫阳性率逐渐增加,均显著高于E15(P<0.05)。PTEN基因的这种表达模式和鸡胚/雏鸡肝脏的发育模式基本一致,说明家禽PTEN基因在肝脏发育和代谢中发挥重要作用。

孵化过程中鸡胚性别分化期的判定

【目的】研究鸡胚孵化过程中,通过不同方法判断其性别分化的时间。【方法】以黄羽肉鸡胚蛋为研究对象,孵化黄羽肉鸡胚蛋(温度37.8℃,相对湿度60%)。分别通过胚线观察法、生殖腺器官形态观察法、组织学形态学、分子生物学来判断鸡的性别分化期,试验重复3次。【结果】通过胚线观察法分析发现,在鸡胚孵化期第3天,可以通过血管分支的粗细和形态来辨别雌雄。通过生殖腺器官的观察,在孵化期第8天,能够明显关察到雄性性腺对称发育,雌性性腺则不对称发育。通过组织学形态鉴定,在孵化期第7~10天,观察到雄性性腺有类似于曲精细管的团索状结构出现,雌性性腺有皮髓结构出现,能够判定鸡胚性别分化期可能为胚胎孵化期第7天。通过分子生物学鉴定,孵化期第3~10天,观察到雄性个体有1条扩增条带,其大小是为550 bp左右,观察到雌性个体有2条扩增条带,其大小为550 bp和450 bp左右,因此判定性别分化期可能为孵化期第3天。【结论】综上判定鸡性别分化期约在胚胎孵化期第3~8天。

Lipid transport to avian oocytes and to the developing embryo

Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of com- parative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipo- protein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen＇s liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. Oocytes grow from a diameter of 5 mm to 2.5-3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. The underlying key transport mechanism is receptor-mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. Recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. As outlined here, lipoprotein degradation/resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the tbllicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo.

鸡胚外泌体对小鼠延缓衰老的作用

研究鸡胚外泌体对小鼠延缓衰老的作用。超高速离心法制备鸡胚外泌体并鉴定;通过体外造血集落形成实验证明其促进造血的能力;用D-半乳糖(D-galactose,D-gal)制作小鼠衰老模型,灌胃不同浓度鸡胚外泌体,检测行为学、血清学指标和肝肾病理等。集落实验显示鸡胚外泌体组的红系爆式集落形成单位(Burst Forming Unit-Erythroid,BFU-E)集落体积更大,低、中、高剂量组的集落数量分别比对照组增加了63.90%、59.47%、68.82%(P<0.01)。与衰老组相比,鸡胚外泌体高剂量组小鼠的胸腺指数、原站台象限停留时间占比、四肢抓力分别升高了71.12%、54.46%(P<0.05)和21.21%(P<0.01),而疲劳仪掉落次数则减少了64.89%(P<0.05)。抗氧化相关指标中,血清总抗氧化能力(Total Antioxidant Capacity,T-AOC)和谷胱甘肽(Glutathione,GSH)的含量分别比衰老组升高了60.41%和396.72%(P<0.01),而丙氨酸氨基转移酶(Alanine Aminotransferase,ALT)降低了27.88%(P<0.05)。病理组织分析显示,鸡胚外泌体低、高剂量组均可有效缓解由D-gal致衰引起的小鼠肝肾损伤。综上,鸡胚外泌体可显著促进造血、改善衰老鼠的脑功能和肌肉力量,减少体内炎症并提高机体抗疲劳及抗氧化能力,改善肝肾等大脏器的结构和功能,从而发挥显著的延缓衰老功能。该文为开发具有延缓衰老功能的新产品提供理论依据。

基于转录组测序挖掘雌性鸡胚性腺不对称发育中的重要lncRNA及其靶基因

为了挖掘雌性鸡胚性腺不对称发育中的重要长链非编码RNA(lncRNA)及其靶基因,试验以采集的胚胎孵化第9天(E9阶段)的雌性鸡胚两侧性腺为研究对象,采用转录组测序(RNA-seq)技术和生物信息学方法对雌性鸡胚左右两侧性腺进行转录组分析预测lncRNA,运用DESeq2 v1.30.1软件鉴定两侧性腺间差异表达的mRNA和lncRNA,预测差异表达lncRNA的顺式和反式靶基因,对靶基因进行GO功能注释和KEGG信号通路分析;通过实时荧光定量PCR扩增验证重要靶基因的表达。结果表明:转录组分析预测出653个基因间lncRNA,467个内含子lncRNA,1339个反义lncRNA。雌性鸡胚左右两侧性腺间有1856个差异表达mRNA和443个差异表达lncRNA。GRIA1、DIO3、FSHR、LHX9、CYP17A1和TOX3等229个基因为差异表达lncRNA的靶基因。两侧性腺差异表达lncRNA的靶基因主要富集到了类固醇代谢过程、细胞内雌激素受体信号通路的正向调节、代谢通路等过程。实时荧光定量PCR扩增验证重要靶基因的表达结果与RNA-seq结果基本一致。说明雌性鸡胚左右两侧性腺具有不同表达水平和特异性表达的mRNA和lncRNA,它们可能影响雌性鸡胚左右两侧性腺的不对称发育。