肝靶向药物载体半乳糖基蛋白的合成及其特性研究

目的：合成肝靶向药物载体半乳糖基人血清白蛋白．并探讨其生物学特性。方法：以半乳糖和人血清白蛋白为原料．合成半乳糖基人血清白蛋白．利用现代生物质谱技术,分析该药物载体的纯度和糖密度;并用^131I标记与模型药物5一氟脲啶偶联后的肝靶向药物载体．进行动物体内靶向性作用的研究。结果：合成的肝靶向药物载体具有较高的纯度,糖密度(即每分子人血清蛋白偶联半乳糖的分子数)达到了51：1．具有显著的趋肝性,肝脏摄取率达到75．0％。结论：合成的肝靶向药物载体半乳糖基人血清白蛋白是优良的受体介导的肝靶向药物载体。     

同源重组敲除MSTN基因的猪胎儿成纤维细胞的构建

 【目的】获得敲除肌肉生长抑制素（MSTN）基因的猪胎儿成纤维细胞。【方法】打靶载体的构建：以Neo为正筛选基因、HSV-tk为负筛选基因。在Neo的两侧分别插入同源长臂和同源短臂。同源长臂5 382 bp,包含MSTN基因的部分5′端,全部的exon1,intron1和exon2及大部分intron2;同源短臂844 bp,包含部分exon3及3′端的部分序列。取35 d胎龄的大白猪,用胰酶消化法,分离胎儿成纤维细胞并对其进行培养和建系。采用脂质体法将打靶载体导入胎儿成纤维细胞中,转染后的细胞采用250 μg?ml-1 G418筛选7 d,再用200 μg?ml-1 G418+2μmol?L-1 GANC维持筛选。用RT-PCR法检测转染前转染后细胞MSTN基因表达量。【结果】成功构建了对猪MSTN基因部分intron2和exon3区域进行敲除的替代型打靶载体。共得到5个具有药物抗性的细胞克隆,经PCR检测,其中一个细胞克隆发生了正确的同源重组。转染后细胞MSTN基因表达量明显降低。【结论】获得了敲除MSTN基因的猪胎儿成纤维细胞。     

多位点基因打靶的定点整合效率研究

利用PCR方法从pEGFP-N1中扩增pCMV-EGFP,从人基因组rDNA基因家族靶基因插入位点两侧分别扩增两条基因打靶同源重组引导序列DS1和DS2,将DS1、DS2片段插入pMD19-pCMV-EGFP中,构建成多位点基因打靶载体pMD19-DS2-pCMV-EGFP-DS1.通过脂质体将其转染至HEK293细胞内.通过荧光检测和PCR、测序等方法,检测和评价定点整合效率.试验结果表明,外源基因EGFP在转染细胞中持续稳定表达,EGFP定点整合率约为4%,不经药物筛选大大提高了整合效率,比传统的基因打靶技术提高了4000多倍,为转基因动物研究建立了高效的定点转基因技术.     

锌指核酸酶技术在基因治疗中的应用研究进展

锌指核酸酶技术是一种新兴的基因高效靶向修饰和调控技术,目前该技术已在人类疾病的基因治疗中得到应用,通过敲除致病基因使其丧失致病性或者通过修复突变基因使基因表达正常,实现了对多种人类疾病的基因治疗。基于提高锌指核酸酶效率、特异性和降低细胞毒性等方面的研究较少的现状,特就人工锌指核酸酶介导的基因敲除和基因同源重组修复等在人类疾病基因治疗方面的应用及存在问题进行了综述,并对相关研究的开展进行了展望。     

Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice

Background

The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.

Findings

Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.

Conclusions

Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.     

Modeling a farm population to estimate on-farm compliance costs and environmental effects of a grassland extensification scheme at the regional scale

We used a farm-level modeling approach to estimate on-farm compliance costs and environmental effects of a grassland extensification scheme in the district of Ostprignitz-Ruppin, Germany. The behavior of the regional farm population (n = 585) consisting of different farm types with different production orientations and grassland types was modeled under the presence and absence of the grassland extensification scheme using the bio-economic model MODAM. Farms were based on available accountancy data and surveyed production data, while information on farm location within the district was derived from a spatial allocation procedure. The reduction in total gross margin per unit area was used to measure on-farm compliance costs. A dimensionless environmental index was used to assess the suitability of the scheme to reduce the risk of nitrate-leaching.Calculated on-farm compliance costs and environmental effects were heterogeneous in space and farm types as a result of different agricultural production and site characteristics. On-farm costs ranged from zero up to almost 1500 Euro/ha. Such high costs occurred only in a very small part of the regional area, whereas the majority of the grassland had low on-farm costs below 50 Euro/ha. Environmental effects were moderate and greater on high-yield than on low-yield grassland. The low effectiveness combined with low on-farm costs in large parts of the region indicates that the scheme is not well targeted. The soft scheme design results from an attempt to achieve environmental and rural development objectives with only one scheme. Improving the efficiency of the scheme would require designing separate instruments for the two distinct objectives. This is in line with the Tinbergen rule, which states that consistent economic policy requires that the number of instruments equals the number of targets.     

簇毛麦3V染色体短臂特异分子标记的开发与应用

普通小麦野生近缘物种簇毛麦(Haynaldia villosa L. 2n=14, VV) 3V染色体短臂（3VS）携带抗小麦条锈病基因。开发高密度的3VS特异标记是准确鉴定和追踪3VS、推进抗病基因转移和利用的重要基础。为了开发更多的均匀分布于3VS染色体臂不同区段的分子标记,本研究利用簇毛麦和小麦品种中国春参考基因组序列与基因注释信息,通过比较3VS与3AS、3BS、3DS同源基因序列内含子序列差异,选择同一内含子长度差异大于10%的区域开发了104个跨越内含子(intron targeting, IT)的分子标记,在簇毛麦、簇毛麦-硬粒小麦双二倍体、硬粒小麦中引1286、中国春、普通小麦-簇毛麦二体异附加系DA1V-DA7V、小麦-簇毛麦T3VS·3DL易位系中进行扩增,筛选出3VS特异、扩增条带清晰且稳定性好的IT分子标记43个。为了验证上述标记的有效性,选择位于3VS不同区段的12个标记对［DS3V(3D)×中国春ph1b］的F₃群体中的148个ph1b纯合的单株进行分析,共筛选得到6种类型涉及3VS的结构变异体。进一步利用GISH/FISH技术对上述材料进行鉴定,结果与分子标记结果相吻合,证明利用外源染色体特异分子标记可有效筛选涉及外源染色体结构变异体。     

New strategy of constructing the β2m gene targeting vector

AIM:The purpose of this study was to establish a new strategy for constructing the mouse β₂m gene targeting vector in order to increase the homologous recombination frequency in contrast with our previous one, which was successfully constructed in the normal way.METHODS:A 4.2 kb 3' arm and a 0.8 kb 5' arm were amplified by PCR from the mouse β₂m-pSV2△HXgpt genomic clone. They included the start region and the three exons, which were separated into two parts from exons 2 (the main coding block) for the two arms——5' arm and 3' arm.RESULTS:The two fragments, in reverse orientation to the Neo gene, were cloned into pPNT respectively on either side of Neo. They were identified by PCR, restriction analysis and sequence analysis as well.CONCLUSION:The mouse β₂m gene targenting vector has been cloned successfully.     

A Rapid DNA Mini-prep Method for Large-Scale Rice Mutant Screening

With the completion of whole genome sequence, focus on rice research has been apparently shifted from sequence analysis (genomics) to identifying and understanding the functions (functional genomics) of nearly 40 000 genes presented in rice genome [1-3]. Gene functional identification requires new materials and methods. Mutant is considered as one of the most important sources of starting materials. In mutant detection, a new reverse genetics strategy termed TILLING (Targeting Induced Loca…