High-throughput fluorescent screening of transgenic animals: phenotyping and haplotyping.

BACKGROUND: Methods for genotyping transgenic animals currently consist of extracting genomic DNA from blood or tissue followed by PCR or Southern blot analysis. These methods when used to screen large numbers of animals can be time consuming and expensive. Therefore, we developed a novel method that allows high-throughput screening of phenotypic changes on leukocytes, resulting from the transgenic genotype. This technique allows investigators to quickly screen a large number of animals without the need to extract DNA from each one. Moreover, since blood is collected for the initial screening, putative homozygotes can be confirmed by conventional methods using the same blood samples. METHODS: We collected blood from wild-type alphagal positive and alphagal knockout mice and probed for the presence of Galalpha(1-->3)Gal (alphagal) epitopes. Also, alloantigen specific antibodies were used to determine the haplotype of our outbred mouse colony in order to develop an inbred line. RESULTS: alphagal epitopes were detected in wild-type but not alphagal knock-out samples. To validate these results, PCR was used to demonstrate the native alphagal gene in wild-type and the pGKneo construct in alphagal knock-out mice. Furthermore, haplotypes were determined and mice divided for backcrosses. CONCLUSIONS: This screening method is useful for both preliminary screening of transgenic mice and the development of an inbred mouse colony by rapid determination of MHC I haplotype. Here, we demonstrate the use of this technique and show how it can be a valuable tool, saving time and resources in both investigator effort and animal husbandry.     

A quantitative competitive PCR method to determine the parasite load in the brain of Toxoplasma gondii-infected mice

Efficacy of vaccine candidates against toxoplasmosis may be expressed in terms of reduction in cyst number in brains of animals vaccinated and then challenged with a cyst-forming strain of Toxoplasma gondii, compared to non-vaccinated animals. Cyst number generally has been determined by microscopic examination of brain homogenate samples, a technique which has a low sensitivity and is time-consuming. Here we describe a quantitative competitive PCR method, which allows quantifying T. gondii DNA in brain samples. The method uses a primer pair, which allows the amplification of a 301 bp fragment of the 35-fold repeated T. gondii B1 gene and an internal standard (non-homologous competitor) derived from phage lambda, which can be amplified using the same primers and whose size and G/C content are similar to that of the B1 target sequence. The method is sensitive (as few as 10 parasites can be quantified), reproducible, and is not affected by the presence of DNA extracted from mouse brain by means of a simple and rapid technique. It is suitable to quantify the parasite load in the brain of infected mice and to evaluate efficacy of toxoplasmosis vaccine candidates.     

Easy and rapid method of zygosity determination in transgenic mice by SYBR<Superscript>®</Superscript> Green real-time quantitative PCR with a simple data analysis

Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.     

High-throughput conformation-sensitive gel electrophoresis for discovery of SNPs

High-throughput screening for single nucleotide polymorphisms (SNPs) or mutations can be achieved by inexpensive technologies. We modified the original protocols of conformation-sensitive gel electrophoresis (CSGE) to increase throughput several fold to 1.3 samples/min, which is about five times faster than denaturing high-performance liquid chromatography (DHPLC). The modifications include decreasing the gel thickness, increasing the number of lanes to 96, and increasing the number of samples per lane to seven. This high-throughput CSGE method is fast, robust, and as simple as the original protocols. Together with a two-stage strategy for screening homozygotes and the replacement of ethidium bromide with SYBR Gold DNA dye staining, this protocol is a reliable and cost-effective alternative for laboratories that require high-throughput screening.     

Detection of tumor mutations in the presence of excess amounts of normal DNA

Mutations are important markers in the early detection of cancer. Clinical specimens such as bodily fluid samples often contain a small percentage of mutated cells in a large background of normal cells. Thus, assays to detect mutations leading to cancer need to be highly sensitive and specific. In addition, they should be possible to carry out in an automated and high-throughput manner to allow large-scale screening. Here we describe a screening method, termed PPEM (PNA-directed PCR, primer extension, MALDI-TOF), that addresses these needs more effectively than do existing methods. DNA samples are first amplified using peptide nucleic acid (PNA)-directed PCR clamping reactions in which mutated DNA is preferentially enriched. The PCR-amplified DNA fragments are then sequenced through primer extension to generate diagnostic products. Finally, mutations are identified using matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This method can detect as few as 3 copies of mutant alleles in the presence of a 10,000-fold excess of normal alleles in a robust and specific manner. In addition, the method can be adapted for simultaneous detection of multiple mutations and is amenable to high-throughput automation.     

Implementation of a PCR assay of Pasteurella pneumotropica to accurately screen for contaminated laboratory mice

The authors implemented a PCR protocol to rapidly screen for Pasteurella pneumotropica and to accurately identify contaminated laboratory mice in a clinical setting. This protocol was implemented in response to a severe outbreak of P. pneumotropica in their animal facility. Although a sentinel program was in place to routinely screen for P. pneumotropica, it was inadequate for the identification of contaminated animals. As a result, several additional strains of mice were contaminated and developed clinical signs of infection. The authors implemented a screening method using PCR with reported primer pairs previously developed to identify the biotype isolates of P. pneumotropica in laboratory mice. Throat culture swabs were collected from live mice and placed in a bacterial culture. The DNA from these cultures was isolated and screened by PCR. This procedure enabled the authors to eliminate P. pneumotropica from several animal housing rooms. The assay can be easily applied in most animal facilities.     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

The use of cortisone-treated mice in the screening of soil for pathogenic fungi

Summary Fifty-seven samples of desert soils were screened for pathogenic fungi by injection into cortisone-treated and non-treated mice. Four samples yielded pathogenic fungi in the cortisone-treated mice only.Nocardia asteroides was isolated from the cortisone-treated mice and from the controls. All of the cortisone-treated and the control mice injected with soil from the root zone of a dead century plant became critically ill very shortly after injection and cultures from all of the animals showed a variety of fungi. Seven species were isolated from the cortisone-treated animals and 5 species from the controls.The yeast,Torulopsis glabrata, was found in 69 of the 225 control mice and in 115 of the 213 cortisone-treated animals that came to autopsy. This organism was not considered to have been derived from the soil.The results appear to justify the conclusion that this method of soil screening deserves further consideration and study.     

DNA detection in hair of transgenic mice--a simple technique minimizing the distress on the animals

The breeding of transgenic animals requires that each individual offspring be analysed for integration of transgenic deoxyribonucleic acid (DNA), unless exclusively homozygous animals are mated. The standard protocol for identification of transgenic animals (Hogan et al. 1994) is based on tissue samples and preparation of chromosomal DNA including proteinase K digestion and phenol/chloroform extraction. The procedure described here represents a much simpler and faster method to screen offspring for the transgene DNA. It is based on the use of hair bulbs as sample material, which can be directly used for polymerase chain reaction (PCR) after alkaline lysis. This protocol allows large numbers of animals to be easily screened in a minimum amount of time. A unique advantage though, is the reduction of the distress caused to the animals. With respect to the 3Rs (Replacement, Reduction, Refinement), and because of technical advantages this method may replace ear or tail clipping.     

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT, EC 2.3.1.28) enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with [14C]chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of 14C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated 14C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intact mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.     

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.     

Feasibility and Recommendations for Swift Fox Fecal DNA Profiling

Abstract: Genetic profiling using fecal samples collected noninvasively in the wild can provide managers with an alternative to live-trapping. However, before embarking on a large-scale survey, feasibility of this methodology should be assessed for the focal species. Costs associated with fecal genotyping can be high because of the need for multiple amplifications to prevent and detect errors. Assessing the relative amount of target DNA before genotyping means samples can be eliminated where error rates will be high or amplification success will be low, thereby reducing costs. We collected fecal samples from an endangered population of swift fox (Vulpes velox) and employed target-DNA quantification and a screening protocol to assess sample quality before genetic profiling. Quantification was critical in identifying samples of low quality (68%, <0.2 ng/μL). Comparison of the amplification, from a subset of loci in 25 samples that did not meet the screening criteria, confirmed the effectiveness of this method. The protocol, however, used a considerable amount of DNA, and an assessment of the locus and sample variability allowed us to refine it for future population surveys. Although we did not use <50% of the samples we collected, the remaining samples provided 36 unique genotypes, which corresponded to approximately 70% of animals estimated to be present in the study area. Although obtaining fecal DNA from small carnivores is challenging, our protocol, including the quantification and qualification of DNA, the selection of markers, and the use of postgenotyping analyses, such as DROPOUT, CAPWIRE, and geographic information, provides a more cost-effective way to produce reliable results. The method we have developed is an informative approach that wildlife managers can use to conduct population surveys where the collection of feces is possible without the need for live-trapping.     

Sensitive and specific molecular detection of Burkholderia pseudomallei, the causative agent of melioidosis, in the soil of tropical northern Australia

Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils.     

一种适用于昆虫痕量DNA模板制备的方法

近年来分子生物学技术在昆虫学各领域中得到了广泛地运用 ,从昆虫样品中有效地获得DNA模板是实验成功的前提。但是由于许多昆虫体形微小 ,许多研究需要取单个个体的样品 ,用传统的酚∶氯仿抽提法难以从痕量样品中获得总DNA ,而某些生物公司的试剂盒相对而言价格昂贵。该文介绍一种快速简便、广泛适用于不同种类昆虫、各种不同保存方法保存的昆虫样品和标本的微量DNA模板制备方法     

一种改进的显微注射用DNA片段的纯化方法

目的显微注射用DNA的纯度是影响转基因动物制备成功与否的重要因素,本文建立一种可行的适用于普通实验室的纯化DNA方法,替代普遍使用的试剂盒纯化方法。方法分别使用酚-氯仿多次抽提法及常规的凝胶提取试剂盒纯化含有蚓激酶基因的DNA片段,通过显微注射技术将纯化的DNA片段导入小鼠受精卵的原核,制备转基因小鼠。根据转基因实验的结果对两种方法进行比较。结果使用两种方法纯化DNA均能获得转基因小鼠。在DNA纯度及注射卵的存活率上,两种方法无明显差别;在移植卵的出生率及转基因阳性率上,抽提法优于试剂盒法。结论本实验建立的抽提方法可以替代试剂盒方法纯化显微注射用DNA片段,在降低实验成本、简化实验条件及提高转基因阳性率方面具有优势。     

Characterizing the reproducibility of a protein profiling method for the analysis of mouse bronchoalveolar lavage fluid

The detection of biomarkers in biological fluids has been advanced by the introduction of mass spectrometry screening methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which enables the detection of the presence and the molecular mass of proteins in unfractionated mixtures. The generation of reproducible mass spectra over the course of an experiment is vital in obtaining data in which differences in protein profiles between diseased and healthy states can be assessed correctly. We have developed a protocol to automate the collection of protein profiling data from a large number of samples using MALDI-TOFMS, and we used these samples to characterize the technical reproducibility of the method. This protocol has been used for the analysis of proteins found in bronchoalveolar lavage fluid samples from mice with the ultimate goal of enabling the discovery of differential expression patterns predictive of the development of chronic obstructive pulmonary disease. Samples were purified using magnetic bead-based technology and analyzed on an AnchorChip target plate. Our results demonstrate that the number of peaks detected reproducibly decreases significantly as sample size increases, which motivates the need for technical replicates to be explicitly included in the analysis of MALDI-TOF-based protein profiling studies.     

A Widely Applicable Protocol for DNA Isolation from Fecal Samples

Feces are increasingly used as sources of DNA for genetic and ecological research. This paper describes a new method for isolation of DNA from animal feces. This method combines multiple purification steps, including pretreatment with ethanol and TE, an inhibitor-absorber made of starch, the CTAB method, the phenol-chloroform extraction method, and the guanidinium thiocyanate-silica method. The new method is efficient according to PCR results of 585 fecal samples from 23 species and costs much less than the commercial kits. The protocol can be tailored to the specific purpose of examining different diets of animals and can be performed with routine laboratory reagents.     

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals (n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.     

High throughput tissue preparation for large-scale genotyping experiments

A fast, efficient technique is described for the extraction of DNA from a large number of samples. The applications of this method include population genetics, plant breeding, and genetic screening. In the field, samples are collected in premeasured silica gel aliquots in polypropylene blocks, which are later used to grind the dried tissue. This permits naturalists, breeders, and collaborators to collect a large number of samples in a short amount of time and allows the samples to dry quickly during shipping. No phenol or chloroform steps are required to obtain high-quality DNA. Samples representing 12 plant families, three invertebrates, and a mammal were included. Quantities of DNA obtained were consistent with or better than other techniques. The quality of samples was tested by amplification of the internal transcribed spacer region. Test amplifications were successful, confirming the quality of extracted DNA.     

Partial uracil–DNA–glycosylase treatment for screening of ancient DNA

The challenge of sequencing ancient DNA has led to the development of specialized laboratory protocols that have focused on reducing contamination and maximizing the number of molecules that are extracted from ancient remains. Despite the fact that success in ancient DNA studies is typically obtained by screening many samples to identify a promising subset, ancient DNA protocols have not, in general, focused on reducing the time required to screen samples. We present an adaptation of a popular ancient library preparation method that makes screening more efficient. First, the DNA extract is treated using a protocol that causes characteristic ancient DNA damage to be restricted to the terminal nucleotides, while nearly eliminating it in the interior of the DNA molecules, allowing a single library to be used both to test for ancient DNA authenticity and to carry out population genetic analysis. Second, the DNA molecules are ligated to a unique pair of barcodes, which eliminates undetected cross-contamination from this step onwards. Third, the barcoded library molecules include incomplete adapters of short length that can increase the specificity of hybridization-based genomic target enrichment. The adapters are completed just before sequencing, so the same DNA library can be used in multiple experiments, and the sequences distinguished. We demonstrate this protocol on 60 ancient human samples.