A rapid and accurate method for herpesviral gnome editing / 生物工程学报

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.

Application of combined chromosomal karyotyping and BACs-on-Beads assay for the prenatal diagnosis of high-risk gravida from Ningbo / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of combined chromosomal karyotyping and BACs-on-Beads(BoBs) assay for the prenatal diagnosis of high risk gravida from Ningbo.</p><p><b>METHODS</b>For 2779 women, results of conventional karyotyping analysis and BoBs assay were compared.</p><p><b>RESULTS</b>For common aneuploidies involving chromosomes 13, 18, 21, X and Y, the two methods have yielded a concordance rate of 98.78%. Eight cases detected with microduplication by BoBs were missed by karyotyping analysis. On the other hand, 17 structural chromosomal abnormalities, 10 chimeras and 1 triploidy detected by karyotyping analysis were missed by BoBs.</p><p><b>CONCLUSION</b>The BoBs technology has featured high throughput and rapidity, and can detect 9 microdeletion syndromes, which can improve the quality of prenatal diagnosis and provide an ideal complementary for conventional chromosomal karyotyping.</p>

Prenatal diagnosis of a tetrasomy 18p case using BACs-on-Beads technology and single nucleotide polymorphism array / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To determine the origin of a supernumerary small marker chromosome found in a fetus using prenatal BACs-on-Beads (BoBs) and single nucleotide polymorphism array (SNP-array) assays.</p><p><b>METHODS</b>The fetal sample was subjected to chromosomal karyotyping and BoBs analysis, and the results were validated with genome-wide scanning using a SNP microarray.</p><p><b>RESULTS</b>The fetus was found to have a 47,XX,+mar karyotype. BoBs analysis indicated that there was an amplification between 18p11.32 and 18p11.21, which was verified by the SNP-array assay as a 18.3 Mb duplication occurring at 18p11.32q11.1.</p><p><b>CONCLUSION</b>The karyotype of the fetus was determined as 47,XX,+der18(18p11.32?18q11.1::18q11.1?18p11.32). The duplication has involved important genes including SMCHD1, LPIN2 and TGIF1, which may result in severe malformations in the fetus.</p>

Application of BoBs technique for detecting common chromosome microdeletion and microduplication syndromes / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a strategy for screening and diagnosing common microdeletion and microduplication syndromes among children with idiopathic mental retardation and development abnormalities.</p><p><b>METHODS</b>Potential chromosomal variations among patients with unexplained mental retardation, cardiac anomalies, particular facial features, learning disabilities and other clinical characteristics were detected with bacterial artificial chromosome BACs-on-Beads (BoBs) technique and karyotyping. Positive results were verified with array-based comparative genomic hybridization (Array-CGH).</p><p><b>RESULTS</b>Fifty eight of the 60 patients had a normal chromosome karyotype. Ten patients with microdeletion and microduplication syndromes were detected by BoBs, which included two positive cases identified through chromosome karyotyping. Two patients were respectively diagnosed as Smith-Magenis syndrome and Prader-Willi/Angelman syndrome by BoBs and the results were confirmed by Array-CGH.</p><p><b>CONCLUSION</b>BoBs is capable of detecting chromosome microdeletion and microduplication with high specificity and throughput, which can compensate the shortcomings of conventional cytogenetic technology and will be widely applied for clinical diagnosis.</p>

Cashmere goat bacterial artificial chromosome recombination and cell transfection system / 生物工程学报

The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination.

Construction and identification of HSV-1 vector vaccine carrying HIV-1 antigen / 生物工程学报

To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.

Exploring the Antarctic soil metagenome as a source of novel cold-adapted enzymes and genetic mobile elements / Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Find and replace: editing human genome in pluripotent stem cells

Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.

Construction of a recombinant BAC-HSV-1 strain HF with a GFP reporter gene and characterization of its infectious progeny virus / 病毒学报

To construct the plasmid of BAC-HSV-1 with GFP reporter gene and research the biological property of its infectious progeny virus. We constructed the plasmid C223-UL43-left-arms-UL47-right-arms which carried the homologous sequences of HSV-1. Liposome embedding method was used to transfect HSV-1 genome and the plasmid C223-UL43-left-arms-UL47-right-arms linearized by Mlu I digestion into Vero cells. After the successful homologous recombination in the eukaryotic cells, the recombinant BAC-HSV-1 with GFP reporter gene was generated. Then, the positive CPE were taken by plaque purification and by hirt extraction during the moment of the circularization of HSV-1 DNA, and the plasmid of BAC -HSV-1 was acquired. Electroporation was used to transfect the BAC -HSV-1 into DH10B, and then the single colonies of interest were confirmed both by MluI digestion and PCR. Experimental group and the control group cells were given BAC-HSV-1 plasmid and HSV-1 genomic DNA respectively to produce the BAC-HSV-1 and HSV-1 progeny virions. Vero cells were inoculated with the progeny virions at MOI = 0.1 and then a TCID50 assay was performed to determine the titers of virons in the two groups at 48 hours post inoculation. The plasmid BAC-HSV-1 was successfully constructed by the restriction enzyme analysis and the PCR. The titers of progeny virions were calculated by the TCID50 assay. No significant difference in the titers of virions between two groups was observed (P > 0.05). The infectious BAC-HSV-1 shuttle virus/plasmid between eukaryotic and prokaryotic cells was successfully constructed.

Seeing Is Believing: Illuminating the Source of In Vivo Interleukin-7

Interleukin-7 (IL-7) is an essential cytokine for T cells. However, IL-7 is not produced by T cells themselves such that T cells are dependent on extrinsic IL-7. In fact, in the absence of IL-7, T cell development in the thymus as well as survival of naive T cells in the periphery is severely impaired. Furthermore, modulating IL-7 availability in vivo either by genetic means or other experimental approaches determines the size, composition and function of the T cell pool. Consequently, understanding IL-7 expression is critical for understanding T cell immunity. Until most recently, however, the spatiotemporal expression of in vivo IL-7 has remained obscured. Shortage of such information was partly due to scarce expression of IL-7 itself but mainly due to the lack of adequate reagents to monitor IL-7 expression in vivo. This situation dramatically changed with a recent rush of four independent studies that describe the generation and characterization of IL-7 reporter mice, all utilizing bacterial artificial chromosome transgene technology. The emerging consensus of these studies confirmed thymic stromal cells as the major producers of IL-7 but also identified IL-7 reporter activities in various peripheral tissues including skin, intestine and lymph nodes. Strikingly, developmental and environmental cues actively modulated IL-7 reporter activities in vivo suggesting that IL-7 regulation might be a new mechanism of shaping T cell development and homeostasis. Collectively, the availability of these new tools opens up new venues to assess unanswered questions in IL-7 biology in T cells and beyond.

Construction of an infectious clone of pseudorabies virus strain ZJ genome maintained as a bacterial artificial chromosome / 病毒学报

pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.

Ectopic Expression of Ephrin-A5 Under the EphA8 Promoter at the Anterior Region of the Superior Colliculus

EphA/ephrin-A mediated signaling has emerged as a key mechanism regulating axon guidance and topographic mapping, particularly in the well-characterized visual system from the retina to the superior colliculus (SC). In this study, EphA8 bacterial artificial chromosome (BAC) was manipulated to contain a floxed eGFP and human ephrin-A5 expression cassette using homologous recombination method. In the mice containing the recombinant BAC, it was shown that GFP is expressed in an anterior>posterior gradient in the SC. Furthermore, when these mice were crossed with the transgenic mice expressing Cre under the EphA8 promoter, it was evident that a GFP expression cassette was eliminated, and that human ephrin-A5 was ectopically expressed in the anterior region of the SC. This transgenic model would be useful to analyze the role of ephrin-A5 in the SC during the retinocollicular topography formation.

Integrated analysis of copy number alteration and RNA expression profiles of cancer using a high-resolution whole-genome oligonucleotide array

Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.

Construction of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome / 生物工程学报

The aim of this study was to construct the complete genome of Marek's disease virus serotype 814 strain as an infectious bacterial artificial chromosome (BAC). Using self-designed selection marker Eco-gpt (1.3 kb) and BAC vector pBeloBAC11 (7.5 kb), we constructed the transfer plasmid pUAB-gpt-BAC11. The plasmid pUAB-gpt-BAC11 and MDV total-DNA were cotransfected into secondary CEFs; we put the virus-containing cells in selection medium for eight rounds and obtained purified recombinant viruses. Recombinant viral genomes were extracted and electroporated into E. coli, BAC clones were identified by restriction enzyme digestion and PCR analysis. Finally, we obtained 38 BAC clones, DNA from various MDV-1 BACs was transfected into CEFs, and recombinant virus was reconstituted by transfection of MDV-BAC2 DNA. We successfully cloned the complete genome of MDV-1814 strain as an infectious bacterial artificial chromosome. With these cloned genomes, a revolutionary MDV-DNA engineering platform utilizing RED/ET recombination system was constructed successfully, which can help the understanding of MDV gene functions and promote the using of MDV as a vector for expressing foreign genes. In addition, it opens the possibility to generate novel MDV-1 vaccines based on the BACs.

Molecular and cytogenetic study on 5 cases with gonadal dysgenesis: clinical applications of fluorescence in situ hybridization(FISH) and BAC-FISH / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the applications of fluorescence in situ hybridization (FISH) in the diagnosis for the patients with gonadal dysgenesis.</p><p><b>METHODS</b>After routine gynecologic examination, ultrasonography and endocrine examination, 5 cases of gonadal dysgenesis and hypogonadism were analyzed by using chromosomal diagnoses including G-banding, Q-banding, multiplex FISH and BAC-FISH analyses.</p><p><b>RESULTS</b>Among the 5 cases of gonad agenesis patients, 2 were pure gonadal dysgenesis with 46, XY karyotype, 3 were mixed gonadal dysgenesis with mos 45, X/47, XXX; 45, X/46, XY or 46, X, der(Y) karyotype.</p><p><b>CONCLUSION</b>Sex chromosomal abnormalities resulted in gonadal dysgenesis symptoms. Applications of FISH and BAC-FISH analyses can correctly diagnose the sex chromosomal abnormalities for patients with gonad agenesis and provide accurate medical genetic data for clinical diagnosis and therapy.</p>

Three-dimensional PCR-based screening of Chinese fine wool merino sheep BAC library / 生物工程学报

For rapid screening, we constructed two levels pools (primary and secondary pools) of the bacterial artificial chromosome (BAC) library of Chinese fine wool merino sheep. The primary pools were based on the individual 384-well microtiter plate and were prepared with a three-dimensional pooling scheme. Three dimension (plate, row and column) pools were made for each. The secondary pools were based on the entire BAC library. We developed a PCR based strategy to identify positive BACs from sheep BAC library. First, we analyzed secondary pools DNAs, according to the result, we analyzed correlative primary pools. It was one-step screening (66 PCR reactions) that we could screen a single positive clone from 74 000 BACs by our method, or three-step screening (less than 100 PCR reactions) could screen more clones. By one-step screening (66 PCR reactions), we screened successfully a positive clone 373D13 with polymorphism marker BF94-1.

Comparative genomics of grasses tolerant to aluminum

The family Poaceae includes over 10,000 species, among which are the most economically important cereals: maize, sorghum, rice, wheat, rye, barley, and oat. These cereals are very important components of human and animal food. Although divergence of the members of this family occurred about 40 million years ago, comparative genome analyses demonstrated that gene orders among species of this family remain largely conserved, which can be very useful for understanding their roles and evolution. Even with an intricate evolutionary history in which chromosome fragments, losses and duplications have to be considered at the ploidy level, grasses present a genetic model system for comparative genomics. The availability of mapped molecular markers, rice genome sequences and BAC and EST libraries from several grass species, such as rice, wheat, sorghum, and maize, facilitates biology and phylogeny studies of this group. The value of using information from different species in modern plant genetics is unquestionable, especially in the study of traits such as tolerance to aluminum in soils, which affects plant growth and development. Comparative genomic approaches to aluminum tolerance can identify genomic regions and genes responsible for aluminum tolerance in grasses.

The study of optimal conditions of electroporation in Escherichia coli DH10B strain / 生物工程学报

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.

Change of the chromosome conformation of human alpha-globin gene locus in transgenic mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To establish chromosome conformation capture (3C) strategy and to use this method for exploring the effect of chromosome conformation on human alpha-globin gene expression in the human alpha-globin transgenic mouse.</p><p><b>METHODS</b>Homozygous human alpha-globin transgenic male mouse was crossed with KM female mouse. The 14.5-day post-coitum (dpc) embryos were used for the isolation of fetal liver and fetal brain cells. Homogeneous single-cell suspension was treated with formaldehyde to crosslink the chromatin conformation in the nuclear. The cross-linked chromatin compound was digested with Nco I and then ligated with T4 DNA ligase. The ligated compound was reversely cross-linked and then the ligated genomic DNA was purified for PCR analysis. The primers were designed along the two sides of cut and ligated sites. Semi-quantitative PCR was used to analyze the chromosome conformation of the whole human alpha-globin gene locus in fetal liver and fetal brain cells.</p><p><b>RESULTS</b>When HS40 fragment was used as the fixed fragment, in fetal brain cells, the ligation frequencies of HS40 fragment with other fragments were decreased as the linear distances to HS40 fragment were increasing; while in fetal liver cells, two active genes (alpha1 and alpha2) fragments showed higher ligation frequencies with HS40 fragment than other fragments. However, the fragment containing an inactive gene (xi) displayed the comparable low ligation frequency as that in fetal brain. When alpha2 fragment was used as the fixed fragment, similarly, in fetal brain cells the ligation frequencies of alpha2 fragment with other ones were decreased as the linear distances increasing; when in fetal liver cells, it showed higher ligation frequencies with two upstream regulatory elements (HS 40 and 33). However, it showed a little bit lower ligation frequency with another two upstream regulatory elements (HS10 and 8) than those in fetal brain.</p><p><b>CONCLUSION</b>In fetal liver cells, the distant regulatory elements are in close proximity to the downstream of the expressed globin genes through looping out, the interval region; however, in fetal brain, they were not in vicinity to the expressed globin genes.</p>