Generation of Amplified RNAs and cDNA Libraries from Single Mammalian Cells

2007 ◽  
pp. 117-124 ◽  
Author(s):  
James Adjaye
Keyword(s):  
Mammalian Cells ◽  
Cdna Libraries

Functional genomics approach to hypoxia signaling

2004 ◽  
Vol 96 (2) ◽  
pp. 765-773 ◽  
Author(s):  
Karen A. Seta ◽  
David E. Millhorn
Keyword(s):  
Cell Survival ◽  
Mammalian Cells ◽  
Cdna Libraries ◽  
Vascular Endothelial ◽  
Pheochromocytoma Cells ◽  
Adaptive Mechanisms ◽  
Endothelial Growth Factor ◽  
Microarray Analyses ◽  
Expressed Sequence ◽  
Sustained Hypoxia

Mammalian cells require a constant supply of oxygen to maintain energy balance, and sustained hypoxia can result in cell death. It is therefore not surprising that sophisticated adaptive mechanisms have evolved that enhance cell survival during hypoxia. During the past few years, there have been a growing number of reports on hypoxia-induced transcription of specific genes. In this review, we describe a unique experimental approach that utilizes focused cDNA libraries coupled to microarray analyses to identify hypoxia-responsive signal transduction pathways and genes that confer the hypoxia-tolerant phenotype. We have used the subtractive suppression hybridization (SSH) method to create a cDNA library enriched in hypoxia-regulated genes in oxygen-sensing pheochromocytoma cells and have used this library to create microarrays that allow us to examine hundreds of genes at a time. This library contains over 300 genes and expressed sequence tags upregulated by hypoxia, including tyrosine hydroxylase, vascular endothelial growth factor, and junB. Hypoxic regulation of these and other genes in the library has been confirmed by microarray, Northern blot, and real-time PCR analyses. Coupling focused SSH libraries with microarray analyses allows one to specifically study genes relevant to a phenotype of interest while reducing much of the biological noise associated with these types of studies. When used in conjunction with high-throughput, dye-based assays for cell survival and apoptosis, this approach offers a rapid method for discovering validated therapeutic targets for the treatment of cardiovascular disease, stroke, and tumors.

scholarly journals Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase

1999 ◽  
Vol 340 (2) ◽  
pp. 561-568 ◽  
Author(s):  
Marc FRANSEN ◽  
Paul P. VAN VELDHOVEN ◽  
Suresh SUBRAMANI
Keyword(s):  
Mammalian Cells ◽  
Unsaturated Fatty Acids ◽  
Urate Oxidase ◽  
Cdna Libraries ◽  
Molecular Evidence ◽  
Cdna Clones ◽  
Novel Proteins ◽  
M13 Phage ◽  
Coa Reductase ◽  
Protein Vi

To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions. In an initial series of biopanning experiments, four different cDNA clones were obtained. These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1). A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms. Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells. The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1. That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein. These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR. In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

cDNA library preparation from total RNA extracts of Single-cell marine protists (e.g. Acantharia, Strombidium basimorphum, and Prymnesium parvum) for transcriptome sequencing v2

2022 ◽  
Author(s):  
Joost S S. Mansour ◽  
Konstantinos Anestis ◽  
Fabrice Not ◽  
Uwe John
Keyword(s):  
Single Cell ◽  
Cdna Library ◽  
Mammalian Cells ◽  
Single Cells ◽  
Rna Extraction ◽  
Cdna Libraries ◽  
Prymnesium Parvum ◽  
Large Variability ◽  
Total Rna ◽  
Marine Protists

Many marine protists are not culturable and therefore challenging to study, nonetheless, they are essential in all marine ecosystems. The development of single-cell techniques is allowing for more marine protists to be studied. Such genomic approaches aim to help to disentangle heterotrophic processes such as phagotrophy from osmotrophy and phototrophic-induced anabolic activities. This information will then support cellular and metabolic modeling by better elucidating the physiological mechanisms and quantifying their importance in different scenarios. However, single-cell protocols and low input RNA kits for transcriptomics are usually made for and tested with mammalian cells, as such the feasibility and efficiency of single-cell transcriptomics on highly diverse mixotrophic protists is not always known. Often single-cell transcriptomics of microbial eukaryotes shows low transcript recovery rates and large variability. We report on transcriptomic methods that we have successfully performed on single cells of Acantharia, Strombidium basimorphum, and Prymnesium parvum. This protocol follows up after total RNA extraction (from the protocol at dx.doi.org/10.17504/protocols.io.bp6xmrfn) to prepare cDNA libraries for Illumina sequencing. The described protocol uses the SMART-Seq4 kit (Takara #634891) for cDNA synthesis and amplification, but this can also be successfully performed with the NEBNext kit (NEB #E6421). The NEBNext kit protocol is very similar to the protocol described here and generally the manufacture's protocol can be followed but see the notes at step 4 and step 18 of this protocol, and do the final elution after cDNA purification in 10 mM Tris (pH 8.0). The subsequent cDNA library is prepared following the .

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells

1985 ◽  
Vol 5 (11) ◽  
pp. 3251-3260
Author(s):  
J D Tratschin ◽  
I L Miller ◽  
M G Smith ◽  
B J Carter
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Viral Vector ◽  
Vector System ◽  
Cdna Libraries ◽  
Foreign Gene ◽  
Dna Transfection ◽  
Adeno Associated Virus ◽  
Hela Cell Lines ◽  
Resistant Cells

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.

scholarly journals An improved protocol for small RNA library construction using High Definition adapters

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Ping Xu ◽  
Martina Billmeier ◽  
Irina Mohorianu ◽  
Darrell Green ◽  
William D Fraser ◽  
...  
Keyword(s):  
Small Rna ◽  
Mammalian Cells ◽  
Leaf Tissue ◽  
Cdna Libraries ◽  
Clinical Samples ◽  
High Definition ◽  
Cdna Insert ◽  
Sequencing Platform ◽  
Ligation Product ◽  
Commercial Kits

AbstractNext generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with greatly reduced proportion of adapter-adapter products due to the degradation of nonligated 3’ adapters. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue.

scholarly journals Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

1995 ◽  
Vol 15 (2) ◽  
pp. 704-710 ◽  
Author(s):  
I Whitehead ◽  
H Kirk ◽  
R Kay
Keyword(s):  
Mammalian Cells ◽  
Retroviral Vectors ◽  
Cdna Libraries ◽  
Expression Cloning ◽  
Cdna Clones ◽  
Beta Catenin ◽  
Functional Screening ◽  
Nucleotide Exchange ◽  
Inhibition Of Proliferation ◽  
Retroviral Transfer

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.

Isolation of novel Src substrates

2003 ◽  
Vol 31 (1) ◽  
pp. 25-28 ◽  
Author(s):  
S.A. Courtneidge
Keyword(s):  
Tyrosine Kinase ◽  
Mammalian Cells ◽  
Protein Production ◽  
Adaptor Protein ◽  
Cdna Libraries ◽  
Novel Genes ◽  
Kinase Substrates ◽  
Proof Of Principle

We have established and used a method to rapidly isolate tyrosine kinase substrates. The method entails inserting mammalian cDNA libraries into phage vectors. Protein production is induced, then plaque proteins are transferred to nitrocellulose and phosphorylated by the kinase of interest. Proof of principle for this technique was established by the isolation of a number of known Src substrates. We also implicated other known proteins as substrates for Src by this approach, and isolated a number of novel genes. Several of these are indeed Src substrates in mammalian cells. We have characterized further one of these novel substrates, Fish, which is a multi-domain adaptor protein.

scholarly journals Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.

1995 ◽  
Vol 15 (5) ◽  
pp. 2612-2624 ◽  
Author(s):  
M Ohtsubo ◽  
A M Theodoras ◽  
J Schumacher ◽  
J M Roberts ◽  
M Pagano
Keyword(s):  
Cell Cycle ◽  
Protein Function ◽  
Mammalian Cells ◽  
Cyclin E ◽  
S Phase ◽  
Cdna Libraries ◽  
Amino Terminal ◽  
E Protein ◽  
Cdk2 Complex ◽  
Alternatively Spliced

Cyclin E was first identified by screening human cDNA libraries for genes that would complement G1 cyclin mutations in Saccharomyces cerevisiae and has subsequently been found to have specific biochemical and physiological properties that are consistent with it performing a G1 function in mammalian cells. Most significantly, the cyclin E-Cdk2 complex is maximally active at the G1/S transition, and overexpression of cyclin E decreases the time it takes the cell to complete G1 and enter S phase. We have now found that mammalian cells express two forms of cyclin E protein which differ from each other by the presence or absence of a 15-amino-acid amino-terminal domain. These proteins are encoded by alternatively spliced mRNAs and are localized to the nucleus during late G1 and early S phase. Fibroblasts engineered to constitutively overexpress either form of cyclin E showed elevated cyclin E-dependent kinase activity and a shortened G1 phase of the cell cycle. The overexpressed cyclin E protein was detected in the nucleus during all cell cycle phases, including G0. Although the cyclin E protein could be overexpressed in quiescent cells, the cyclin E-Cdk2 complex was inactive. It was not activated until 6 to 8 h after readdition of serum, 4 h earlier than the endogenous cyclin E-Cdk2. This premature activation of cyclin E-Cdk2 was consistent with the extent of G1 shortening caused by cyclin E overexpression. Microinjection of affinity-purified anti-cyclin E antibodies during G1 inhibited entry into S phase, whereas microinjection performed near the G1/S transition was ineffective. These results demonstrate that cyclin E is necessary for entry into S phase. Moreover, we found that cyclin E, in contrast to cyclin D1, was required for the G1/S transition even in cells lacking retinoblastoma protein function. Therefore, cyclins E and D1 control two different transitions within the human cell cycle.

scholarly journals Isolation of a Mammalian Homologue of a Fission Yeast Differentiation Regulator

1999 ◽  
Vol 19 (5) ◽  
pp. 3829-3841 ◽  
Author(s):  
Hanako Yamamoto ◽  
Kappei Tsukahara ◽  
Yoshihide Kanaoka ◽  
Shigeki Jinno ◽  
Hiroto Okayama
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Erythroid Differentiation ◽  
Cdna Libraries ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Human Leukemia ◽  
Amino Acid Homology ◽  
Binding Domains

ABSTRACT In the fission yeast Schizosaccharomyces pombe thenrd1 + gene encoding an RNA binding protein negatively regulates the onset of differentiation. Its biological role is to block differentiation by repressing a subset of the Ste11-regulated genes essential for conjugation and meiosis until the cells reach a critical level of nutrient starvation. By using the phenotypic suppression of the S. pombetemperature-sensitive pat1 mutant that commits lethal haploid meiosis at the restrictive temperature, we have clonedROD1, a functional homologue ofnrd1 +, from rat and human cDNA libraries. Likenrd1 +, ROD1 encodes a protein with four repeats of typical RNA binding domains, though its amino acid homology to Nrd1 is limited. When expressed in the fission yeast,ROD1 behaves in a way that is functionally similar tonrd1 +, being able to repress Ste11-regulated genes and to inhibit conjugation upon overexpression. ROD1is predominantly expressed in hematopoietic cells or organs of adult and embryonic rat. Like nrd1 + for fission yeast differentiation, overexpressed ROD1 effectively blocks both 12-O-tetradecanoyl phorbol-13-acetate-induced megakaryocytic and sodium butyrate-induced erythroid differentiation of the K562 human leukemia cells without affecting their proliferative ability. These results suggest a role for ROD1 in differentiation control in mammalian cells. We discuss the possibility that a differentiation control system found in the fission yeast might well be conserved in more complex organisms, including mammals.

Sign in / Sign up

Export Citation Format

Share Document