Nuclear transfer of goat somatic cells transgenic for human lactoferrin gene

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins with appropri-ate post-translational modifications.The nuclear transfer of transgenic somatic cells is a powerful method to pro-duce mammary gland bioreactors.We established an effi-cient gene transfer and nuclear transfer approach in goat somatic cells.Gene targeting vector pGBC2LF was con-structed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene and the endogenous start codon was replaced by that of human LF gene.Goat fetal fibroblasts were transfected with lin-earized pGBC2LF and 14 cell lines were positive accord-ing to PCR and Southern blot.The transgenic cells were used as donor cells of nuclear transfer and some of recon-structed embryos could develop into blastocyst in vitro.     

整合人lactoferrin基因的山羊体细胞支持核移植克隆胚的体外发育

克隆了人lactoferrin基因和山羊[[beta]]-casein基因5′端调控区, 构建了人lactoferrin的乳腺表达载体, 并将该载体利用脂质体介导转染了奶山羊胎儿成纤维细胞, 获得了稳定整合人lactoferrin基因的转基因体细胞克隆17个, 其中PCR和Southern Blot检测阳性的细胞克隆14个, 阳性率82.4%。以转基因体细胞为供体细胞进行了核移植, 获得了能够体外发育的山羊转基因克隆胚胎, 体内成熟卵母细胞来源的核移植囊胚率为64.8%, 体外成熟卵母细胞来源的核移植囊胚率为51.7%, 证明了山羊转基因体细胞能够支持克隆胚的进一步发育。     

Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production

Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.     

以经过转染的乳腺上皮细胞生产克隆羊

为研究转基因乳腺上皮细胞发育的全能性,利用电转染方法将人乳铁蛋白(hLF)乳腺特异性表达载体电转染山羊乳腺上皮细胞,经G418和PCR筛选获得阳性克隆细胞株,经催乳素诱导的细胞株上清液用Western blotting方法检测hLF的表达。以转基因与上清液中表达hLF均为阳性的细胞为核供体细胞,进行山羊体细胞核移植。结果为:16株细胞表达重组hLF,分子质量为75 kD;将144枚重构胚移入16只同步发情的山羊输卵管中,在移植后的30 d、60 d和90 d的妊娠率分别为87.5%、81.3%和62.5%;最终3只受体妊娠足月,产下3只克隆羊,克隆效率为2.1%,PCR-RFLP分析表明克隆羊均来自供体羊细胞,但没有整合外源基因。结果表明,hLF转基因乳腺上皮细胞能分泌hLF;乳腺上皮细胞经转染、筛选和长期培养的条件下,能保持发育的全能性。     

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.     

转录激活因子样效应物核酸酶介导的山羊β-乳球蛋白基因敲除和人乳铁蛋白基因定点整合

为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF~+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF~+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF~+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。     

单、双标记基因筛选的转人乳铁蛋白基因供核细胞生产克隆山羊效率的比较

为比较两种筛选标记基因生产转人乳铁蛋白(hLF)基因克隆山羊的效率,利用单(新霉素抗性基因,Neor)、双(新霉素抗性和绿色荧光蛋白基因,Neor/GFP)标记基因筛选转基因的供核细胞,并制作体细胞核移植转基因山羊。山羊胎儿成纤维细胞电转染单标记基因表达载体(pBLC14)或双标记基因表达载体(pAPLM),分别有58.8%(20/34)和86.7%(26/30)的抗性细胞株检测到外源基因;转染pAPLM的细胞传代培养后,仅有20%(6/30)株细胞在传代中所有细胞均能观察到荧光;分别以pBLC14和pAPLM的细胞株作为供核细胞进行体细胞核移植,共获得806枚重构胚胎,胚胎移植受体后35 d、60 d妊娠率分别为53.8%、26.9%和39.1%、21.7%,最终分别产下5只(1.9%)和7只(1.4%)克隆山羊;经PCR及Southern blotting检测,所有出生山羊均整合有外源基因。结果显示,以单、双标记基因筛选供核细胞,其重构胚融合率、怀孕率和克隆动物出生率差异不显著(P>0.05),Neor/GFP双标记基因能准确、有效地用于转基因供核细胞筛选。同时,结果也表明Neor/GFP双标记基因转染的体细胞作为供核细胞对体细胞克隆效率未出现不利影响。     

体细胞基因打靶制备动物乳腺生物反应器的策略与应用

在转基因动物研究中,由于基因表达调控元件的人工拼接和外源基因在动物基因组中随机整合所带来的“位置效应”,致使转基因动物外源基因的表达水平不高并且差异较大。为此,利用定位整合优势,对以基因同源重组为基础的基因打靶技术进行了大量研究。介绍了就利用体细胞基因打靶和核移植技术制备动物乳腺生物反应器的策略和应用情况做一综述,并对提高基因打靶效率的各种策略,打靶细胞的选择,转基因细胞核移植的低融合事件以及基因打靶制备乳腺生物反应器的优越性进行分析。     

利用多重筛选机制提高山羊乳腺上皮细胞基因打靶的效率

构建了山羊β-酪蛋白基因座基因打靶载体pGBC—GFP—neo,载体包含了正负筛选标记基因neo和tk,以及无启动子的GFP基因。打靶载体线性化后用脂质体包裹转染山羊乳腺上皮细胞,利用G418和GANC进行抗性细胞克隆的药物筛选,共得到抗性细胞克隆51个,对抗性克隆利用激素诱导β-酪蛋白基因启动子指导GFP表达,得到GFP表达阳性细胞克隆17个,对其中4个生长状态良好的克隆PCR检测验证后用于核移植,克隆胚发育率为59．5％,部分发育到桑椹胚或囊胚。     

Expression of active recombinant human lactoferrin in the milk of transgenic goats

This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats.     

Polymorphic sequence of Korean Native goat lactoferrin exhibiting greater antibacterial activity

Lactoferrin, which exhibits antibacterial activity to protect infants from infectious disease, is a major component of colostrum and milk. Lactoferrin was purified from the colostrum of Korean Native goat, and the cDNA from the mammary gland mRNA of the animal was cloned and sequenced. The nucleotide sequence of the lactoferrin gene of Korean Native goat was found to differ in 15 sites from that of the goat lactoferrin gene reported earlier. This difference in nucleotide sequence resulted in six amino acid substitutions: five in the N-lobe and one in the C-lobe. The antibacterial activity of Korean Native goat lactoferrin was found to be greater than that of Sannen goat lactoferrin.     

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.     

Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 g ml^–1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.     

奶山羊乳腺上皮细胞的分离、培养及鉴定

应用组织块培养法高密度培养、连续传代法建立西农萨能奶山羊乳腺上皮细胞体外培养体系,通过生长曲线绘制、核型分析、免疫荧光染色 (角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白)、油红染色及β-酪蛋白基因的RT-PCR分析进行培养细胞鉴定。实验结果表明细胞生长曲线为典型的S型,染色体数目众数为60,细胞角蛋白、上皮膜抗原、波形蛋白、β-酪蛋白表达均呈阳性,油红染色后可见细胞质内的脂滴,且细胞表达酪蛋白mRNA。说明运用本方法培养的细胞为正常的乳腺上皮细胞,并具有一定的泌乳功能。     

动物乳腺生物反应器研究进展

利用转基因家畜的乳腺生产人类重组蛋白,可以高效获得安全、足量的药用蛋白。本在简要介绍乳腺生物反应器的基本原理及优越性的基础上,对其目的基因、表达载体和转基因技术在国内外的研究现状加以综述,着重探讨了体细胞核移植方法生产乳腺生物反应器的优越性及面临的技术问题。     

Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer

Summary The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.     

ht-Pam基因在山羊β-酪蛋白基因座定位整合的研究

利用体细胞基因打靶与核移植技术制备动物乳腺生物反应器是当今转基因定位整合表达的一种新技术。分别克隆山羊的β-酪蛋白基因5′调控区的6.3kb片段,外显子7、外显子8和9三个基因片段,并与克隆的人tPA突变体cDNA一起构建了含有neo和tk正负筛选标记基因的β-酪蛋白基因打靶载体P^GBC4tPA,并验证了neo基因、tk基因以及Cre-LoxP系统的有效性。将线性化的P^GBC4tPA通过电转染整合到山羊胎儿成纤维细胞基因组中,利用G418和GANC进行抗性细胞克隆的药物筛选,初步获得抗性细胞克隆244个,PCR检测后获得阳性细胞克隆31个,其中初步验证2个细胞克隆转植基因整合位点重组后的基因序列正确,并且该细胞克隆能够有效扩增。这为下一步基因打靶体细胞核移植制备山羊乳腺生物反应器奠定了基础。     

Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex-pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans-genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cash-mere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats.     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.