Transfection of insect cell lines using polyethylenimine

Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been frequently done using labor intensive and cytotoxic liposome-based transfection reagents. In the current study we have optimized a new kind of polyethylenimine-based DNA transfection reagent on the Spodoptera frugiperda Sf9 insect cell line. A plasmid vector that transiently expresses green fluorescent protein (GFP) was effectively delivered into Sf9 cells. A transfection efficiency of 54% and cell viability of 85–90% were obtained for Sf9 cells. The developed transfection protocol has now been successfully used to transfect eight insect cell lines derived from Bombyx mori, Trichoplusia ni, Helicoverpa zea, Heliothis virescens and S. frugiperda with GFP and GUS with transfection efficiencies of at least 45%. This method provides high heterologous protein expression levels, transfection efficacy and cell viability, and could be used for transient gene expression in other lepidopteran cell lines.

Genetic distance of inbred lines and prediction of maize single-cross performance using RAPD markers

 To evaluate the genetic diversity of 18 maize inbred lines, and to determine the correlation between genetic distance and single-cross hybrid performance, we have used random amplified polymorphic DNA (RAPD), a PCR-based technique. Eight of these lines came from a Thai synthetic population (BR-105), and the others derived from a Brazilian composite population (BR-106). Thirty two different primers were used giving a total of 325 reproducible amplification products, 262 of them being polymorphic. Genetic divergence was determinated using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method with arithmetical averages (UPGMA). Cluster analysis divided the samples into three distinct groups (GI, GII and GIII) that were confirmed by principal-coordinate analysis. The genetic distances (GD) were correlated with important agronomic traits for single-cross hybrids and heterosis. No correlation was found when group division was not considered, but significant correlations were detected between GI×GII and GI×GIII GDs with their respective single-cross hybrid grain-yield values. Three groups were identified; that is, the BR-106 population was divided in two different groups and the BR-105 population remained mostly as one group. The results indicated that RAPD can be used as a tool for determining the extent of genetic diversity among tropical maize inbred lines, for allocating genotypes into different groups, and also to aid in the choice of the superior crosses to be made among maize inbred lines, so reducing the number of crosses required under field evaluation. Received: 24 May 1996 / Accepted: 22 November 1996     

Parental analysis using RAPD markers in the antColobopsis nipponicus: A test of RAPD markers for estimating reproductive structure within social insect colonies

Summary Previously known parent-offspring relationship for queens and her daughters of the antColobopsis nipponicus was examined using RAPD markers in order to test the reliability of this molecular technique for estimating the reproductive structure within colonies of social insects. RAPD markers from 20 oligomers successfully clustered the queen with her daughters among an artificially generated polygynous society, even when paternal information was unavailable. When information from both the queen and her sperm was included in the analysis, 20 polymorphic bands seem to be sufficient to cluster correctly the true parents to their offspring. Lack of father's information considerably decreased accuracy of the analysis. Thus, if RAPD markers are to be used to demonstrate parent-offspring relationship between individuals in the field, sperm from the queen's spermatheca should be incorporated in analysis.     

Identification of Bactrian camel cell lines using genetic markers

Iranian Bactrian camel population is less than 100 animals. Iranian biological resource center produced more than 50 Bactrian camel fibroblast cell lines as a somatic cell bank for conservation animal genetic resources. We compared two type markers performance, including 14 random amplified polymorphic DNA (RAPDs) (dominant) and eight microsatellite (co-dominant) for cell line identification, individual identification and investigation genetic structure of these samples. Based on clarity, polymorphism, and repeatability, four RAPD primers were selected for future analysis. Four RAPD primers and eight microsatellite markers have generated a total of 21 fragments and 45 alleles, respectively. RAPD primers revealed fragment size between 150 to 2000 bp and gene diversity since 0.27 (IBRD) to 0.46 (GC10), with an average of 0.37. Microsatellite markers generated number of alleles per locus ranged from 3 to 11, with an average of 5.62 alleles. The observed heterozygosity ranged from 0.359 (IBRC02) to 0.978 (YWLL08), and expected heterozygosity ranged from 0.449 (IBRC02) to 0.879 (YWLL08). Bottleneck analysis and curve showed that Bactrian camel population did not experience a low diversity. RAPD profiles were especially suitable for investigation population genetics. All primers generated novel and polymorphic fragments. Briefly, our results show that a multiplex PCR based on these markers can still be valuable and suitable for authentication of cell lines, investigating gene diversity and conservation genetic resources in Bactrian camel, while new technologies are continuously developed.     

Development and characterization of insect cell lines

Conclusions With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the midgut, it may not be possible to obtain cell lines from these tissues from all insect species due to terminal differentiation and other factors. Also, researchers have desired cell lines from certain species, such as the honey bee, for which no success has been obtained. As in the early days of tissue culture, it is difficult to discern why negative results occur. However, as more is learned about the physiology and nutrition of various insects and tissues, we may get clues which will help solve these questions.The remaining chapters in this book will provide the reader with exciting uses for insect cell culture. As I mentioned earlier, the baculovirus expression vector system has provided a stimulus to the field of insect cell culture not seen previously.Abbreviations ICD Isocitrate dehydrogenase - ME malic enzyme - PGI phosphoglucose isomerase - PGM phosphoglucose mutase     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata, andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institutes of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749 from the National Institutes of Health.     

Species identity of insect cell lines

Summary Three mosquito cell cultures designated as Suitor's clone ofAedes aegypti, Culiseta inornata andAedes vexans were shown to be moth by immunological, karyological, and isozyme analyses. The cells reacted with rabbit antimoth serum but not rabbit antimosquito serum. Chromosome analyses indicated Lepidopteran rather than Dipteran morphology, and three isozyme systems were confirmative. Any one of these assays would be sufficient to indicate that contamination had occurred and could be used as a periodic check for identity of cell cultures. Morphology and growth characteristics are also valid criteria to distinguish between these particular orders of insect cells. These studies were supported by Grant CA-04953-12 from the National Cancer Institute; General Research Support Grant FR-5582 from the National Institues of Health; and Grant-in-Aid Contract M-43 from the State of New Jersey. Recipient of Research Career Award 5-K3-16,749. from the National Institutes of Health.     

Deterministic paternity exclusion using RAPD markers

The Random Amplified Polymorphic DNA (RAPD) technique can potentially provide hundreds of polymorphic markers for use by ecologists studying mating systems in natural populations. We consider here the implications of the dominance displayed by RAPD markers for deterministic paternity assignment. Our goal was to provide a means for assessing the costs associated with such a study for ecologists who might be considering the use of RAPD markers for paternity analysis. The theoretical expected proportion of offspring for which all males except the true father can be exlucded (P(ET)) is calculated for both dominant and codominant marker systems. The ability to assign paternity unambiguously generally increases with the number of loci and the frequency of the recessive allele (but only up to a point), and decreases with increasing sample size (number of individuals surveyed). The gain in P(ET) with decreasing sample size is unexpectedly slight. Not surprisingly, the performance of dominant markers at paternity exclusion is, in general, greatly exceeded by codominant markers, with the exception of the case in which the frequency of the recessive allele is high at all loci. In this case, codominant markers perform only slightly better than do dominant markers. Thus, a researcher should expect to score more than 50 RAPD loci for each offspring for most applications of paternity exclusion analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of apple cultivars using RAPD markers

Summary Eleven apple cultivars were differentiated using randomly amplified polymorphic DNA (RAPD) markers obtained by the polymerase chain reaction (PCR). The variability of the technique and of the origin of the DNA extract was analyzed. A set of bands consistent in their presence or absence was chosen to create a differentiating band pattern. A key is proposed by which one can differentiate apple cultivars using commercially available prime.     

Rapid authentication of animal cell lines using pyrolysis mass spectrometry and auto-associative artificial neural networks

Summary Pyrolysis mass spectrometry (PyMS) was used to produce biochemical fingerprints from replicate frozen cell cultures of mouse macrophage hybridoma 2C11-12, human leukaemia K562, baby hamster kidney BHK 21/C13, and mouse tumour BW-O, and a fresh culture of Chinese hamster ovary CHO cells. The dimensionality of these data was reduced by the unsupervised feature extraction pattern recognition technique of auto-associative neural networks. The clusters observed were compared with the groups obtained from the more conventional statistical approaches of hierarchical cluster analysis. It was observed that frozen and fresh cell line cultures gave very different pyrolysis mass spectra. When only the frozen animal cells were analysed by PyMS, auto-associative artificial neural networks (ANNs) were employed to discriminate between them successfully. Furthermore, very similar classifications were observed when the same spectral data were analysed using hierarchical cluster analysis. We demonstrate that this approach can detect the contamination of cell lines with low numbers of bacteria and fungi; this approach could plausibly be extended for the rapid detection of mycoplasma infection in animal cell lines. The major advantages that PyMS offers over more conventional methods used to type cell lines and to screen for microbial infection, such as DNA fingerprinting, are its speed, sensitivity and the ability to analyse hundreds of samples per day. We conclude that the combination of PyMS and ANNs can provide a rapid and accurate discriminatory technique for the authentication of animal cell line cultures.     

昆虫细胞系的培养和建立技术

迄今已经报道的昆虫细胞系有800株以上。昆虫细胞系在昆虫病理学、寄生虫学、内分泌学、遗传学和分子生物学等基础和应用研究中得到越来越广泛的应用。本文结合我们研究的结果和实践经验,概括了国内外昆虫细胞系建立技术的研究进展,包括昆虫细胞培养的发展、昆虫细胞系建立技术、不同昆虫组织来源细胞系的建立方法和过程,以及对昆虫细胞系特征的鉴定等方面。     

O-glycosylation potential of lepidopteran insect cell lines

The enzyme activities involved in O-glycosylation have been studied in three insect cell lines, Spodoptera frugiperda (Sf-9), Mamestra brassicae (Mb) and Trichoplusia ni (Tn) cultured in two different serum-free media. The structural features of O-glycoproteins in these insect cells were investigated using a panel of lectins and the glycosyltransferase activities involved in O-glycan biosynthesis of insect cells were measured (i.e., UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, UDP-Gal:core-1 beta1, 3-galactosyltransferase, CMP-NeuAc:Galbeta1-3GalNAc alpha2, 3-sialyltransferase, and UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase activities). First, we show that O-glycosylation potential depends on cell type. All three lepidopteran cell lines express GalNAcalpha-O-Ser/Thr antigen, which is recognized by soy bean agglutinin and reflects high UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity. Capillary electrophoresis and mass spectrometry studies revealed the presence of at least two different UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases in these insect cells. Only some O-linked GalNAc residues are further processed by the addition of beta1,3-linked Gal residues to form T-antigen, as shown by the binding of peanut agglutinin. This reflects relative low levels of UDP-Gal:core-1 beta1,3-galactosyltransferase in insect cells, as compared to those observed in mammalian control cells. In addition, we detected strong binding of Bandeiraea simplicifolia lectin-I isolectin B4 to Mamestra brassicae endogenous glycoproteins, which suggests a high activity of a UDP-Gal:Galbeta1-3GalNAc alpha1, 4-galactosyltransferase. This explains the absence of PNA binding to Mamestra brassicae glycoproteins. Furthermore, our results substantiated that there is no sialyltransferase activity and, therefore, no terminal sialic acid production by these cell lines. Finally, we found that the culture medium influences the O-glycosylation potential of each cell line.     

Comparison of Thoroughbred and Arabian horses using RAPD markers

We compared pools of DNA from 10 Thoroughbred horses and 10 Arabian horses for the presence of randomly amplified polymorphic DNA (RAPD) markers which might be useful in distinguishing between the breeds. Using 212 decamer oligonucleotides and our polymerase chain reaction (PCR) conditions, 173 of the primers produced scoreable bands. The number of bands ranged from 0 to 9 with an average of 3·6. In family studies using 11 arbitrarily selected primers, five of the 11 primers produced polymorphic bands which exhibited Mendelian inheritance as dominant markers. When comparing the pooled DNA from Thoroughbred and Arabian horses we found 10 primers which identified markers present in the pooled DNA from one breed but absent in the pool from the other breed. Testing individual horses revealed that only two markers were wholly absent for one group while being present among members of the other. Primer UBC-85 (5′-GTGCTCGTGC-3′) detected a pair of markers absent in Thoroughbred horses but present among 11 of 31 Arabian horses. These markers were 1500 and 1700 base pairs (bp) long and designated UBC-85^C and UBC-85^D, respectively. Primer UBC-126 (5′-CTTTCGTGCT-3′) detected a 1000 bp marker (designated UBC-126^C) absent in 20 of 20 Thoroughbred horses but present in 31 of 31 Arabian horses. UBC-126^C would be particularly effective for breed comparisons, especially if the DNA band were cloned, sequenced and an allelic marker present in Thoroughbred horses but rare or absent among Arabian horses was identified. The distribution of such markers among other horse breeds might be useful to infer relationships among breeds. These kinds of markers may also be useful in detecting unwanted crossbreeding between two horse breeds.     

Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers

A series of crosses designed for introgression of mandarin (Citrus reticulata Blanco) and pummelo (C. maxima Merr.) germplasm, to develop an alternative rootstock to sour orange (C. aurantium L.), were carried out. It is necessary to identify those hybrids that yield nucellar seedlings for rootstock propagation. Rootstocks can be developed through traditional plant breeding methods; however, the ability to screen and select for economically important traits (such as production of true nucellar seedlings) in an efficient fashion is limited by the difficulties of screening techniques based on whole plant performance. To address these problems, we have used randomly amplified polymorphic DNA (RAPD) and fluorescently labeled expressed sequence tag simple sequence repeat (EST-SSR) molecular markers. A total of 204 individual seedlings obtained from 34 hybrid parental plants were successfully characterized using five RAPD primers. Ten hybrid parents and their progenies, found to be genetically similar among themselves, were selected for more scrutiny using eight EST-SSR primer pairs. The degrees of genetic similarity (nucellars) among progeny seedlings were determined and compared with that of their parents. The mean genetic similarity varied from 67–99% among the selected rootstock candidates screened. The genetic similarity relationship identified using RAPD and EST-SSR molecular markers was highly concordant (p = 0.001). Two elite rootstock candidates (B6R5T56; B6R11T129) that seem to be ideal for future mandarin and pummelo derived rootstock breeding programs have been identified. Our results indicate that either RAPD or EST-SSR analyses could be equally successful in identifying true nucellars among the progenies obtained from introgression crosses of mandarin and pummelo, thus improving the accuracy of early selection in a citrus rootstock breeding program.     

沙田柚系列遗传关系的RAPD标记研究

采用 RAPD标记技术分析了沙田柚系列 1 2个样品的遗传关系。利用经筛选具多态性的 1 4个 1 0碱基随机引物对 1 2个样品进行 DNA随机扩增 ,获得清晰可重复的位点 99个 ,其中多态性位点 5 8个 ,占 5 8.89% ;在部分样品中检出了特异性 RAPD标记 1 9个 ,占 1 9.1 9% ;通过样品间遗传相似性系数比较与 UPGMA聚类分析 ,并根据历史事实可知 ,广西沙田柚、梅州金柚为沙田柚的无性繁殖系 ,软枝系沙田柚、沙田柚早熟单株为沙田柚的芽变后代 ,梅花早柚、菊花心沙田柚、冬瓜圈沙田柚、段氏柚、垫江沙田柚、古老钱沙田柚为沙田柚的实生变异品系。     

Characterization of rainbow trout cell lines using microsatellite DNA profiling

Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. This revised version was published online in August 2006 with corrections to the Cover Date.     

Aphid-symbiotic bacteria cultured in insect cell lines

The cells and tissues of many aphids contain bacteria known as "secondary symbionts," which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names "Candidatus Consessoris aphidicola" for T type and "Candidatus Adiaceo aphidicola" for U type.     

Identification of white truffle species using RAPD markers

Morphologically very similar species of white truffle were analyzed by the random amplified polymorphic DNA (RAPD) technique. Species-specific RAPD fragments were selected and pairs of primers were designed on their sequences. Sequence-characterized amplified regions were developed and applied to identify these species throughout their entire life cycle: fruit body, mycelium, ectomycorrhiza. This procedure provides an unambiguous and rapid tool for typing species whose morphology is very similar.