Rapid cloning of mammalian cells with honeycomb cloning plates and nonlethal vital stains

Summary A rapid and technically simple method for cloning both adhesive and nonadhesive mammalian cells is described. The procedure employs (a) honeycomb cloning plates and (b) nonlethal vital stains. Instead of placing cloning rings around colonies, cells are initially seeded at clonal density directly into a plate containing an array of cloning rings (the honeycomb plate). Hence, the time involved in placing cloning rings around colonies is eliminated. Second, clone-containing wells of the honeycomb plate are easily identified by staining plates with the nonlethal vital stains, MTT or INT tetrazolium. Vital staining eliminates the time involved in searching for clones. Last, clones are transferred with a cotton-tipped swab thereby eliminating the time involved in trypsinization of cells. In this fashion, one can pick and transfer clones ofsubstrate adherent mammalian cells at a rate of one clone/ 10 to 15 s. Thus, mammalian cells can be cloned as rapidly as cloning can be carried out in microbial systems. This study was supported, in part, by Grant CA 33074 from the National Cancer Institute, Bethesda, MD, Grant PCM-8218137 from the National Science Foundation, Washington, D.C., and a grant from the National March of Dimes.     

Improved protocols for the isolation and in-situ cryopreservation of cell colonies

Stable transfection and cloning of cells often require physical separation of cell colonies. In order to conveniently isolate cell clones from petri dishes, we developed a protocol starting with a soft agar overlay of cells. This reduces the risk of cell diffusion between different colonies. Cells from individual colonies are mechanically removed, incubated with trypsin, and cell suspensions are seeded onto parallel microtiter plates. The cell clones on one microtiter plate can be cryopreserved in situ using the protocol described here which was tested for a variety of cell lines. Replica plates can be used for screening and further expansion of interesting clones. If screening can also be performed in situ, e.g., by immunocytochemistry, immunofluorescence, or the polymerase chain reaction, it is possible to perform most steps necessary in cell cloning experiments on microtiter plates.     

Colony formation of mammalian cells on agar plates and its application to Lederberg's replica plating

In this paper we describe a new technique of cloning by use of agar plates and its application to replica plating. It was found that most cell lines form colonies on the surface of solid agar, although the plating efficiency and size of colony is dependent on specimens and concentrations of agar and agarose used. When 0.5% Noble-agar was used as substrate, plating efficiencies were obtained comparable to those of conventional cloning techniques in liquid medium and of agar suspension cultures. In some cases, including the primary culture of Yoshida sarcoma, the efficiency of plating was apparently higher than that obtained by the already established procedures. In an experiment with a series of BHK-21 cells, it was found that virally transformed cells could form colonies on agar plate, whereas untransformed and reverted cells could not divide, suggesting that agar plate culture, as well as agar suspension culture, can be used for a selective assay of transformation.Two methods of replica plating were employed. Method I is that devised by Lederberg in which colonies on the master plate are imprinted on pile fabrics and then transferred to the replica plates. With FM3A cells, the fidelity of replica plating was around 95%. Method II is inoculation of clones by applying a glass rod to the replica plates on which positions of inocula were identified by a grid. Fidelity of replica plating of FM3A, L5178Y and YSC cells was 99.7, 100 and 100% respectively.     

Development of a Method for Detection of Lactic Acid Bacteria Producing Exclusively the l-(+)- Isomer of Lactic Acid

A method was developed for the detection and isolation, within a population of lactic acid bacteria, of strains producing exclusively the l-(+)- isomer of lactic acid; the visual detection of colonies of these particular strains can be carried out directly on agar plates (50 to 70 colonies per plate). The method is based on an enzymatic stereospecific reaction involving d-(-)-lactate dehydrogenase and linked to a staining reaction; the diffusion area of the d-(-)- isomer stains red around the d-(-)- and the dl-lactic acid-producing colonies, while the colonies producing exclusively l-(+)-lactic acid are detected by the absence of the colored halo. The intensity of staining was increased when cellulose powder and Tween 20 were added to the agar medium.     

Development of a novel Bacillus subtilis cloning system employing its neutral protease as screen marker

Part of the pUC19 polylinker sequence (33 bp) was inserted into the pro-peptide-coding region of the Bacillus subtilis neutral protease-encoding gene to replace a 93-bp FspI-HindIII fragment. This in-frame sequence replacement had little effect on the expression and secretion of the neutral protease. This plasmid can therefore be used as a cloning vector, and recombinant clones can be directly identified on skim milk indicator plates by the loss of a clear ring (or halo) around the colonies. This novel cloning system offers several advantages over existing B. subtilis cloning vectors: (i) convenient direct screening of recombinants; (ii) the use of inexpensive indicator; (iii) no restriction on the use of host strains; and (iv) the availability of seven frequently used unique cloning sites: BamHI, XbaI, SalI, PstI, SphI, HindIII, and EcoRI. This system also has the potential to be used as an expression/secretion vector.     

Molecular cloning of a Bacillus subtilis xylanase gene in Escherichia coli

A gene coding for xylanase synthesis in Bacillus subtilis was isolated by direct shotgun cloning using Escherichia coli as a host. Following partial digestion of B. subtilis chromosomal DNA with PstI or EcoRI restriction enzymes, fragments ranging from 3 to 7 kb were introduced into the PstI or EcoRI sites of pBR325. Transformed colonies having lost either the ampicillin or chloramphenicol resistance markers were screened directly on 1% xylan plates. Out of 8000 transformants, ten xylanase-positive clones were identified by the clearing zone around lysozyme-treated colonies. Further characterization of one of the clones showed that the xylanase gene was present in a 3.9-kb insert within the PstI site of the plasmid pBR325. Retransformation of E. coli strain with the xylanase-positive hybrid plasmid pRH271 showed 100% transformation to xylanase production. The intracellular xylanase produced by the transformed E. coli was purified by ion exchange and gel permeation chromatography. The electrophoretic mobility of the purified xylanase indicated an Mr of 22 000.     

Expression cloning systems.

This review will cover the use of expression cloning in Xenopus oocytes, fission yeast, and mammalian cells. Of the systems covered herein, transient expression cloning systems in Xenopus oocytes and mammalian cells have proven to be the most effective and versatile, as demonstrated by the large number of cDNA clones isolated by these two methods in the past year. Of particular interest, are recent advances in the screening methodologies used in conjunction with transient expression in mammalian cells which have permitted the application of this system in the isolation of cDNAs encoding intracellular proteins.     

构建定向T载体用于基因克隆和表达

传统的T载体克隆方法需要烦琐的后续步骤来筛选和鉴定重组子,并且无法实现目的基因的定向克隆。为了克服这些问题,本研究在pET-23a(+)的基础上构建了定向T载体pETG,首先通过定点诱变消除pET-23a(+)上的两个BfuⅠ位点得到PET-23aM;设计一对引物在5端各引入一个BfuⅠ位点,下游引物紧邻BfuⅠ位点引入13 bp的部分LacO序列,用该引物从pHBM2002上扩增Prrn-gfp表达盒,插入PET-23aM的NdeⅠ和XhoⅠ位点,得到定向T载体pETG。PCR扩增的目的基因通过下游引物引入7 bp剩余的LacO序列,该基因片段与BfuⅠ酶切制备的定向T载体连接、转化大肠杆菌DH10β感受态细胞,通过补加了X-gal的平板筛选蓝色重组子。质粒酶切和PCR鉴定表明蓝色菌落全部为定向插入的重组子,重组效率100%,利用本方法成功地定向克隆了103个人类肝蛋白编码基因cDNA,克隆过程无需复杂的步骤筛选鉴定重组子。随机选择了其中的8个基因的克隆进行表达,结果显示8个克隆均在大肠杆菌中获得成功表达。该结果表明定向T载体构建成功,并且该载体非常适合基因的克隆和表达。     

The golden anniversary of cloning: a celebratory essay

May 2002 marked the golden anniversary of the first cloned tadpoles. We celebrate this anniversary, as nuclear transplantation of frog cells into enucleated eggs became the prototype for cloning insects, fish, and mammals. We briefly review the salient results from amphibian cloning. Extension of these studies to mammalian species led to cloning adult cells, important advances in understanding nuclear reprogramming, and the construction of transgenic clones for biomedical applications. In addition, murine cloning clarified two problems unresolved in frog cloning: the unequivocal demonstration that nuclei of fully differentiated cells can direct the formation of fertile adults, and that abnormal expression of genes was responsible for the endoderm and neural syndromes in Rana clones.     

An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I–mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.     

The possible role of cell cycle stage in mammalian cloning

Progress in mammalian cloning started from cloning embryos (of mice, rats, rabbits, sheep, goats, pigs, cattle and rhesus monkeys) and culminated in obtaining clones of sheep, cattle, pigs and mice from adult somatic cells. Knowing the relationship between the cell cycles of the recipient and the donor of cell nucleus in embryonic cloning by nuclear transfer one can adjust the phases of the cell cycle properly. Metaphase II recipients accept G1 (in most species) or G2 donors (in the mouse). Interphase recipients can harbour nuclei in all stages of cell cycle. Relatively little is known about somatic cloning. Two attitudes are applied: either the donor is in the G0 phase or the recipient is in a prolonged MII phase.     

A novel cloning system for direct screening using a suicidal strategy

The pAC92 plasmid is a direct screening cloning vector which allows positive selection of recombinant clones (re-clones). This new high-copy-number plasmid vector encodes ampicillin resistance and carries the Bacillus subtilis α-amylase (α-Amy)-encoding gene (amy) containing a multiple cloning site. The pAC92 plasmid confers to Escherichia coli transformants an amylolytic phenotype easily detected by iodine vapor staining. The re-clones are identified by insertional inactivation of α-Amy activity. During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the promoter region that caused the increase in the amy expression. This suicide characteristic permitted the positive selection of re-clones. A second transformation step was performed to enhance the rate of re-clones per plate.     

A Replica Plating Method for Plant Cells

A replica plating method is described for plant cells growing in Petri dishes. The method involved a uniform application of plant cells (Morinda citrifolia L.) by spraying cells evenly on agar plates containing 60% conditioned medium. Subsequently the cells were allowed to grow through a nylon net. The net was removed from the master plate and placed upside down on replica plates. Cells from colonies adhering to the threads of the net were thus transferred to the replica plate and yielded colonies that, after a growth period of about 10–20 days, corresponded in position to the colonies on the master plate. An 80% transfer of colonies from the master plate to the copy plate was possible.     

A simple and fast method for cloning and analyzing polymerase chain reaction products

A method describing a fast and efficient way for cloning polymerase chain reaction (PCR) products is presented that involves end repair and purification of the PCR product, followed by kinasing and ligation to the vector with the use of a temperature gradient. Efficiency of ligation was estimated to be 50%–70%. Following transformation, cells are plated on MacConkey agar. Bacteria from selected colonies are used directly from the plates for screening without any subsequent purification. Using this protocol, PCR products can be efficiently cloned quickly and economically.     

Microimmunofluorescence using Terasaki plates and direct plate freezing method--rapid and reliable screening system of hybridomas

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at -80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly-L-lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.     

Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.     

A simple technique for reducing edge effect in cell-based assays

Several factors are known to increase the noise and variability of cell-based assays used for high-throughput screening. In particular, edge effects can result in an unacceptably high plate rejection rate in screening runs. In an effort to minimize these variations, the authors analyzed a number of factors that could contribute to edge effects in cell-based assays. They found that pre-incubation of newly seeded plates in ambient conditions (air at room temperature) resulted in even distribution of the cells in each well. In contrast, when newly seeded plates were placed directly in the CO(2) incubator, an uneven distribution of cells occurred in wells around the plate periphery, resulting in increased edge effect. Here, the authors show that the simple, inexpensive approach of incubating newly seeded plates at room temperature before placing them in a 37 degrees C CO(2) incubator yields a significant reduction in edge effect.     

Protoplast fusion in microtiter plates for expression cloning in mammalian cells: demonstration of feasibility using membrane-bound alkaline phosphatase as a reporter enzyme

Protoplast fusion is a method for directly transferring cloned DNA from bacteria to mammalian cells at high efficiency. Here, we have used membrane-bound alkaline phosphatase as a reporter enzyme in a miniprotoplast fusion assay. This work demonstrates the principle that large numbers of protoplast fusions can be done simultaneously and successfully, to assay for an activity encoded by an expression vector. The technique described here circumvents key hurdles to expression cloning. This method does not require a highly sensitive assay or a way of separating a rare expressing cell from the mixture of transfected cells containing other transfected genes. With a strong promoter, the protein encoded by the undiluted transfected cDNA should be produced at at least as high a level as it is endogenously produced in the cell from which its activity was first detected. Reference clones are stored, avoiding the need to separate out the cells that are successfully transfected; this also avoids the need to repurify the DNA from the transfected cell. Because of the use of microtiter plates, it is likely that such a method could be partially automated for many types of assays.     

Porphyrin rings and phospholipids: stimulators of cloning efficiency in certain species of Tetrahymena.

Species of Tetrahymena, including T. vorax, T. thermophila, T. pyriformis, and T. pigmentosa, were tested for cloning efficiency in proteose peptone and in synthetic nutrient media to which were added hemin, protoporphyrin IX, chlorophyllin, or asolectin, an impure mixture of phospholipids. All species could be cloned with high efficiency in the crude media. In unsupplemented synthetic medium the cloning efficiencies were 0-10%, around 50%, around 50%, and 90-100% for T. thermophila, T. vorax, T. pyriformis, and T. pigmentosa, respectively. The first three were all stimulated to 90-100% by addition of the porphyrin or phospholipid compounds mentioned above. Uroporphyrin III and coproporphyrin I and III had no effect. We suggest that cells unable to form clones suffer from a lack of cellular energy. This situation may be alleviated by our additions, certain porphyrin rings may be built into cytochromes and phospholipids may be used as fuel. Thus, the synthetic media used so far for these ciliates have not been optimal.