In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.     

慢病毒介导的牛生长素基因对小鼠胚胎发育的研究

生长素是促进动物生长发育所必需的激素之一。本研究探索牛生长激素(bovine growth hormone,bGH)对小鼠胚胎发育的影响。将牛生长激素基因连接到慢病毒载体(Lenti-CMV-EF1-eGFP)形成Lenti-CMV-bGH/EF1-eGFP,重组病毒经293T细胞包装后直接感染小鼠胚胎和G1胚胎干细胞(embryonicstemcells,ESCs)。阳性表达的胚胎经体外培养,发育到囊胚期的胚胎进行胚胎移植,为直接感染组。感染的小鼠G1胚胎干细胞注射到正常昆明白小鼠囊胚制作嵌合胚胎后进行胚胎移植,为嵌合体组。分别解剖3只妊娠15d的直接感染组和嵌合组的小鼠,观察受体小鼠妊娠情况。结果发现,在1-细胞期感染胚胎,感染效率可达(74.7±6.7)%,在2-细胞期连续感染,感染效率为(79.4±5.7)%,两组感染方法的效率没有显著差异(P>0.05)。1次和2次感染胚胎发育和正常胚胎之间没有显著差异((87.6±3.5)%,(85.4±6.3)%VS(83.0±5.5)%,P>0.05)。所有移植的胚胎没有发育到足月,解剖妊娠15d的小鼠,发现直接感染组没有妊娠,嵌合组胎盘发育正常,但胚胎部分已经死亡和被吸收。因此,本研究证明bGH对小鼠附着前胚胎发育没有影响,但影响小鼠胚胎附着后的发育。这为后期bGH的研究提供参考。     

A New Family of Retroviral Long Terminal Repeat Elements in the Human Genome Identified by Their Homologies to an Element 5′ to the Spider Monkey Haptoglobin Gene

A new family of retroviral long terminal repeats that we name Spm-LTR has been identified as a result of DNA sequence comparisons between the entire GenBank databank and an element, SPHP, located 5′ to the haptoglobin gene of spider monkeys. The 18 human Spm-LTR sequences so identified fall into three subtypes. There is no sequence similarity between Spm-LTR elements and any endogenous retroviral LTR sequences previously reported except for general features that define LTRs. However, a previously described repeated sequence (MER-4) forms a portion of the Spm-LTR sequence.     

HIV—1长末端重复序列对重组痘苗病毒表达Gag蛋白的影响

将系列缺失的ＨＩＶ１长末端重复序列（ＬＴＲ）和全长的ｇａｇＯＲＦ置于痘苗病毒载体中,经同源重组和血球吸附试验,成功地构建了６株重组痘苗病毒。免疫印迹和免疫酶试验检测均表明,６株重组病毒的Ｇａｇ蛋白表达量因ＬＴＲ不同而有明显差异,表明ＨＩＶ１的ＬＴＲ及其下游基因置于痘病毒启动子控制下,在痘苗病毒中表达时有下述特点：（１）不同的痘苗病毒启动子与全长ＬＴＲ相互作用,对ｇａｇ基因表达有显著不同的调控效果;（２）ＮＲ序列对Ｇａｇ蛋白表达没有明显影响;（３）ＥＮ序列不能被重组痘苗病毒表达系统识别;（４）ＴＡＲ序列可提高Ｇａｇ蛋白的表达量;（５）Ｕ５区及下游非翻译序列不影响Ｇａｇ蛋白的表达。     

Optimization of the Transductional Efficiency of Lentiviral Vectors: Effect of Sera and Polycations

Lentiviral vectors are widely used as effective gene-delivery vehicles. Optimization of the conditions for efficient lentiviral transduction is of a high importance for a variety of research applications. Presence of positively charged polycations reduces the electrostatic repulsion forces between a negatively charged cell and an approaching enveloped lentiviral particle resulting in an increase in the transduction efficiency. Although a variety of polycations are commonly used to enhance the transduction with retroviruses, the relative effect of various types of polycations on the efficiency of transduction and on the potential bias in the determination of titer of lentiviral vectors is not fully understood. Here, we present data suggesting that DEAE-dextran provides superior results in enhancing lentiviral transduction of most tested cell lines and primary cell cultures. Specific type and source of serum affects the efficiency of transduction of target cell populations. Non-specific binding of enhanced green fluorescent protein (EGFP)-containing membrane aggregates in the presence of DEAE-dextran does not significantly affect the determination of the titer of EGFP-expressing lentiviral vectors. In conclusion, various polycations and types of sera should be tested when optimizing lentiviral transduction of target cell populations.     

Sequence of the Long Terminal Repeat and Adjacent Segments of the Endogenous Avian Virus Rous-Associated Virus 0

Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U(3) region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U(3) region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U(3) is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.     

Effect of an Insertional Mutation in the cspA Gene Encoding the Major Cold-Shock Protein on Radiation Resistance of Escherichia coli

Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E. coli cells (cspA ⁺). The radiation protective effect of this plasmid is abolished or drastically reduced in mutants recA13and rpoH15defective in RecA protein and in induction of the heat-shock protein regulon, respectively. Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to -irradiation of E. coli mutants with an intermediate level of radiation resistance (Gam^r445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gam^r444 mutant. In the chromosome of E. coli strain with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of -rays and UV light 2.9 and 1.4 times, respectively. These data suggest that the system of cold-shock proteins can modulate resistance of E. colicells to the lethal effect of -rays and UV light.