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Lesson One（4学时）Inside the Living Cell: Structure andFunction of Internal Cell PartsCytoplasm: The Dynamic, Mobile Factory细胞质：动力工厂Most of the properties we associate with life are properties of the cytoplasm. Much of the mass of a cell consists of this semifluid substance, which is bounded on the outside by the plasma membrane. Organelles are suspended within it, supported by the filamentous network of the cytoskeleton. Dissolved in the cytoplasmic fluid are nutrients, ions, soluble proteins, and other materials needed for cell functioning.生命的大部分特征表现在细胞质的特征上。
细胞质大部分由半流体物质组成，并由细胞膜（原生质膜）包被。
细胞器悬浮在其中，并由丝状的细胞骨架支撑。
细胞质中溶解了大量的营养物质，离子，可溶蛋白以及维持细胞生理需求的其它物质。
The Nucleus: Information Central（细胞核：信息中心）The eukaryotic cell nucleus is the largest organelle and houses the genetic material (DNA) on chromosomes. (In prokaryotes the hereditary material is found in the nucleoid.) The nucleus also contains one or two organelles-the nucleoli-that play a role in cell division. A pore-perforated sac called the nuclear envelope separates the nucleus and its contents from the cytoplasm. Small molecules can pass through the nuclear envelope, but larger molecules such as mRNA and ribosomes must enter and exit via the pores.真核细胞的细胞核是最大的细胞器，细胞核对染色体组有保护作用（原核细胞的遗传物质存在于拟核中）。
有关微生物转化的英文文献Title: Microbial Transformation: A Versatile Tool in Biotechnology.Abstract:Microbial transformation, a biotechnological process employing microorganisms to convert one compound into another, has gained significant attention in recent years. This article delves into the principles, applications, and advancements of microbial transformation, discussing itsrole in drug discovery, biofuel production, and environmental remediation.Introduction:Microbial transformation, a subset of biotransformation, refers to the enzymatic or chemical alteration of a compound by microorganisms. These microorganisms, oftenfungi or bacteria, possess unique metabolic capabilitiesthat allow them to convert a wide range of compounds into valuable products. The process is environmentally friendly, sustainable, and often more economical than synthetic methods.Applications of Microbial Transformation:1. Drug Discovery: Microbial transformation has been extensively used in drug discovery and development. Through the biotransformation of naturally occurring compounds, researchers can generate a library of analogs with diverse biological activities. These analogs can then be screened for their pharmacological properties, leading to the identification of potential drug candidates.2. Biofuel Production: Microbial transformation is also being explored for the production of sustainable biofuels. By utilizing lignocellulosic biomass, microorganisms can convert these complex carbohydrates into simple sugars, which can further be fermented into ethanol or other biofuels. This process has the potential to reduce our reliance on fossil fuels and mitigate the impact of climatechange.3. Environmental Remediation: Microbial transformation can play a crucial role in environmental remediation. By using specific microorganisms, pollutants such as hydrocarbons, pesticides, and heavy metals can be degraded or transformed into less toxic compounds. This technology has the potential to clean up contaminated sites and restore ecological balance.Advancements in Microbial Transformation:Recent advancements in microbial transformation have focused on improving the efficiency and selectivity of the process. Genetic engineering techniques have been employed to create microorganisms with enhanced metabolic capabilities. These genetically modified organisms (GMOs) can produce higher yields of desired products or convert a broader range of substrates.In addition, high-throughput screening methods have been developed to rapidly identify microorganisms withdesirable transformation activities. These methods involve the use of automated instrumentation and advanced analytics, enabling the rapid evaluation of large libraries of microorganisms.Conclusion:Microbial transformation, a versatile tool in biotechnology, offers a sustainable and economical alternative to synthetic methods. Its applications in drug discovery, biofuel production, and environmentalremediation demonstrate its wide-ranging potential. With continued advancements in genetic engineering and high-throughput screening, microbial transformation is poised to play an increasingly important role in biotechnology.。
Chapter 5An Efficient Protocol for VZV BAC-Based MutagenesisZhen Zhang, Ying Huang, and Hua ZhuAbstractVaricella-zoster virus (VZV) causes both varicella (chicken pox) and herpes zoster (shingles). As a member of the human herpesvirus family, VZV contains a large 125-kb DNA genome, encoding 70 unique open reading frames (ORFs). The genetic study of VZV has been hindered by the large size of viral genome, and thus the functions of the majority of these ORFs remain unclear. Recently, an efficient protocol has been developed based on a luciferase-containing VZV bacteria artificial chromosome (BAC) system to rapidly isolate and study VZV ORF deletion mutants.Key words:Varicella-zoster virus, Bacterial artificial chromosome, Deletion mutagenesis, Bioluminescence1. I ntroductionVaricella-zoster virus (VZV) is a common human herpesvirus thatis a significant pathogen in the United States, with more than 90%of the US population harboring the virus (1). Primary infectionof VZV leads to varicella (chicken pox). VZV establishes lifelonglatency in the host, specifically in trigeminal ganglia and dorsalroot ganglia (2). The VZV reactivation results in herpes zoster(shingles), which often leads to chronic postherpetic neuralgia(2, 3). As a member of human alpha-herpesvirus subfamily, VZVhas a 125-kb long double-stranded DNA genome, which encodesat least 70 unique open reading frames (ORFs). The genomes ofseveral different VZV strains were sequenced and a few of theVZV genes genetically analyzed (4).It has been extremely difficult to generate VZV site-specificmutations using conventional homology recombination meth-ods. This was mainly due to the high cell-associated nature ofVZV infection in vitro, which leads to the difficulties in isolatingJeff Braman (ed.), In Vitro Mutagenesis Protocols: Third Edition,Methods in Molecular Biology, vol. 634,DOI 10.1007/978-1-60761-652-8_5, © Springer Science+Business Media, LLC 20107576Zhang, Huang, and Zhuviral DNA and purifying recombinant virus away from wild-type virus. In the last few years, a popular method for VZV in vitro mutagenesis involves a four-cosmid system covering the entire viral genome (5–7). Using the cosmid system to generate recom-binant VZV variants involves technically challenging steps such as co-transfection of four large cosmids into permissive mammalian cells and multiple homologous recombination events within a single cell to reconstruct a full-length viral genome. The highly cell-associated nature of VZV also makes the downstream appli-cations of traditional virology methods such as plaque assay-based titering and plaque purification difficult. To date, the functions of the majority of VZV ORFs remain uncharacterized (8).In order to create recombinants of VZV more efficiently, the full-length VZV (P-Oka strain, a cloned clinical isolate of VZV) genome has been successfully cloned as a VZV bacteria artificial chromosome (BAC) (9, 10). This VZV BAC combined with a highly efficiently E. coli homologous recombination system allows quick and easy generation of recombinant VZV. To further ease the downstream virus quantification assays, a firefly luciferase reporter gene, was inserted into the VZV BAC to generate a novel luciferase-expressing VZV (10). In this protocol, we show the generation and analyses of VZV full-length ORF deletion mutants and genetic revertants as examples to demonstrate the utility and efficiency of this versatile system for VZV mutagenesis in vitro. Furthermore, this protocol can be easily modified to broaden its applications to a variety of genetic maneuvers including making double ORF deletions, partial ORF deletions, insertions, and point mutations.1. Human melanoma (MeWo) cells were grown in DMEM supplemented with 10% fetal bovine serum, 100 U penicillin–streptomycin/ml, and2.5 m g amphotericin B/ml at 37°C in a humidified incubator with 5% CO 2. All tissue culture reagents were obtained from Sigma (St. Louis, MO).2. VZV luc was recently developed in the laboratory (10). It con-tains a full-length VZV P-Oka genome with a firefly luciferase cassette (see Note 1). The BAC vector was inserted between VZV ORF60 and ORF61, which includes a green fluorescent protein (GFP) expression cassette and a chloramphenicol resistance cassette (Cm R ).3. pGEM-oriV/kan was previously constructed (11) in the lab-oratory and used as a PCR template to generate the expres-sion cassettes for the kanamycin or ampicillin resistance genes (Kan R and Amp R ).2. M aterials2.1. Cells, VZV luc , Plasmids, and E. coliStrain77An Efficient Protocol for VZV BAC-Based Mutagenesis 4. pGEM-lox-zeo was derived from pGEM-T (Promega, Madison, WI) (12) and was used to generate the rescue clones of VZV ORF deletion mutants. 5. E. coli strain DY380 was obtained from Neal Copeland and Craig Stranthdee and used for mutagenesis (13). 6. A cre recombinase expression plasmid pGS403 was a gift from L. Enquist (Princeton University, NJ). 1. All primers were synthesized by Sigma-Genosys (Woodlands, TX) and stored in TE buffer (100 m M). 2. HotStar Taq DNA polymerase (Qiagen, Valencia, CA) was used for PCR reactions and Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) could be used for optional hi-fidelity PCR reactions (see Note 2). 3. PCR purification was carried out using a PCR purification kit (Qiagen, Valencia, CA). 4. The amplified linear DNAs were suspended in sterile ddH 2O and w ere q uantified b y s pectroscopy (NanoDrop T echnologies, Wilmington, DE). 5. DpnI (New England Biolabs, Ipswich, MA) restriction treat-ment following PCR was carried out in order to eliminate circular template DNA. 6. Electroporation was carried out with a Gene Pulser II Electroporator (Bio-Rad, Hercules, CA). 1. All antibiotics were obtained from Sigma (St. Louis, MO). LB plates containing specific antibiotics were used for appro-priate selections (Table 1).2. A 37°C air shaker and a 37°C water bath shaker were used for bacterial culturing.2.2. Primers, PCR, PCRpurification, DpnITreatment, andElectroporation2.3. AntibioticsSelection and BACDNA Purification Table 1Antibiotics concentrations for selectionFor BACs (single or low copy numbers)For plasmids (high copynumbers)78Zhang, Huang, and Zhu3. NucleoBond Xtra Maxi Plasmid DNA purification kits (Clontech Laboratories, Inc., Palo Alto, CA) were used to purify VZV BAC DNA from E. coli .4. Kimwipes (Kimberly-Clark Global Sales, Inc., Roswell, GA) were used as small filters in BAC DNA Mini-preparations.5. Phenol/chloroform, isopropanol, and ethanol were obtained from Sigma (St. Louis, MO) and were used as additional reagents in BAC DNA preparations.6. Hin dIII (New England Biolabs, Ipswich, MA) digestions were performed to check the integrity of BAC DNA.1. FuGene6 transfection kit (Roche, Indianapolis, IN) was used for transfecting viral BAC DNA into MeWo cells (ATCC).2. An inverted fluorescent microscope was used to observe and count plaque numbers.3. Tissue culture media containing 150 m g/ml d -luciferin (Xenogen, Alameda, CA) was used as substrate for in vitro bioluminescence detection.4. An IVIS Imaging 50 System (Xenogen, Alameda, CA) was used to record bioluminescence signal from virally infected cells.5. Bioluminescence data were quantified by using Living Image analysis software (Xenogen, Alameda, CA).In order to generate VZV ORF deletion mutants using this new VZV luc system, we took advantage of an efficient recombination system for chromosome engineering in E. coli DY380 strain (13). A defective lambda prophage supplies the function that protects and recombines linear DNA. This system is highly efficient and allows recombination between homologies as short as 40 bp. The experimental design is summarized in Fig. 1.1. The first step in making any specific VZV ORF deletion was to amplify a Kan R cassette containing 40-bp flanking sequences of the targeted ORF.2. Primers were stored in TE buffer (100 m M). The Kan R expres-sion cassette was amplified from pGEM-oriV/Kan using a HotStar DNA polymerase kit following a standard protocol.3. PCR product was purified using a PCR purification kit fol-lowing the manufacturer’s protocol.2.4. Transfectionand SubsequentVirological Assays(Tittering and Growth Curve Analysis)3. M ethods3.1. Generation of VZVORF Deletion BACClones 3.1.1. Making a Kan R Cassette Targeting a Specific VZV Open Reading Frame79An Efficient Protocol for VZV BAC-Based Mutagenesis 4. The purified PCR product was treated with DpnI in order to eliminate the template DNA. This step greatly reduces the background in later selections.5. PCR product was purified again as above (step 3) and the amplified linear DNA was suspended in sterile ddH 2O and was quantified by spectroscopy (see Note 3).1. DY380 cells were grown at 32°C until the OD 600nm measure-ment reached 0.5 (see Note 5).2. The culture was shifted to 42°C by placing the flask into a 42°C water bath with vigorous shaking for 10–15 min (see Note 4).3.1.2. Induction of theLambda RecombinationSystem and Preparationof Electroporation-Competent DY380Fig. 1. Generating ORF deletion mutants (ORFD). (a ) The E. coli DY380 strain provides a highly efficient homologous recombination system, which allows recombination of homologous sequences as short as 40 bp. The homologous recombination system is strictly regulated by a temperature-sensitive repressor, which permits transient switching on by incubation at 42°C for 15 min. VZV luc BAC DNA is introduced into DY380 by electroporation. Electro-competent cells are prepared with homologous recombination system activation. (b ) Amplification of the Kan R expression cassette by PCR using a primer pair adding 40-bp homologies flanking ORFX. (c ) About 200 ng of above PCR product are transformed into DY380 carrying the VZV luc BAC via electroporation. (d ) Homologous recombination between upstream and downstream homologies of ORFX replaces ORFX with the Kan R cassette, creating the ORFX deletion VZV clone. The recombinants are selected on LB agar plates containing kanamycin at 32°C. (e ) The deletion of ORFX is confirmed by testing antibiotic sensitivity and PCR analysis. The integrity of viral genome after homologous recombination is examined by restriction enzyme Hin dIII digestion. (f ) VZV luc BAC DNA with ORFX deletion is propagated and isolated from DY380. (g ) Purified BAC DNA is transfected into MeWo cells. (h ) 3–5 days after transfection, the ORFX deletion virus is visualized under a fluores-cent microscope due to EGFP expression given nonessentiality of ORFX.Select for kan Rat 32°Cseqs. (40 bp)ORFX kan R ORFE. coli 32°C ts λ cI repressorVZV-BAC Defective l prophage D BkanR E BAC DNATransfect MeWo cells ProducerecombinantVZV (givenORFX is notessential)x G Hx MR WTORFXDORFXR Confirm recombinant VZV by antibiotic sensitivity, PCR and HindIII digestion80Zhang, Huang, and Zhu3. The culture was immediately transferred to an ice–water slurry for 30 min. (see Note 6).4. After incubation on ice, the culture was then pelleted at 6,000 × g for 10 min 4°C, washed with ice-cold sterile ddH 2O, and repelleted.5. Prechilled 10% glycerol (use about 1% of original volume of culture) was used to resuspend cells, and a 40-m l aliquot (>1 × 1010 cells) was used for each electroporation reaction. 1. Two microliters of Kan R cassette DNA (greater than 200 ng) were electroporated into competent DY380 cells harboring the VZV luc BAC. Homologous recombination took place between the 40-bp ORF flanking sequences and the targeted BAC ORF was replaced by the linear Kan R cassette creating the expected VZV ORF deletion clones. 2. Electroporation was carried out at 1.6 kV, 200 W , and 25 m F in a Gene Pulser II electroporator. Two microliters of con-centrated linear DNA cassette (greater than 200 ng) were used in each reaction. 3. The bacteria were immediately transferred to 1 ml LB medium after electroporation and incubated at 32°C for 1 h before plating. The resultant recombinants were selected on LB agar plates containing kanamycin at 32°C for 16–24 h (see Note 7). 4. Antibiotic sensitivity: it is important to further test that kanamycin-resistant colonies are resistant to kanamycin but not to ampicillin because the circular pGEM-oriV/Kan R (containing Amp R ) was used as the PCR template. This can be tested by re-streaking single colonies on mul-tiple LB agar plates containing different antibiotics. VZV ORFX deletion clones should be resistant to chloram-phenicol (from BAC vector), hygromycin (from luciferase cassette), and kanamycin (VZV ORF replacement cassette), but sensitive to ampicillin (potentially from pGEM-oriV/Kan R ; see Note 8). 1. Mini-BAC DNA preparations.(a) A single DY380 clone containing the recombinant VZV BAC was inoculated in 5 ml LB supplemented with the appropriate antibiotics and cultured at 32°C overnight.(b) BAC DNA was isolated by pelleting the bacteria, resus-pending in 1 ml resuspension buffer supplemented with RNase A (Buffer RES), lysing in 1 ml NaOH/SDS lysis buffer (Buffer LYS), and neutralizing in 1 ml potassium acetate neutralization buffer (Buffer NEU) for 5 min for each step (NucleoBond Xtra Maxi Plasmid DNA purifi-cation kit).3.1.3. Electroporation andRecombinant Screening3.1.4. BAC DNAPurification and BACClone Verification81An Efficient Protocol for VZV BAC-Based Mutagenesis (c) The cloudy solution was centrifuged at 4,500 × g for 15 min at 4°C. The supernatant was filtered through a small piece (cut to 4 × 4 cm) of Kimwipe tissue (Kimberly-Clark Global Sales, Inc., Roswell, GA).(d) The filtered solution was extracted with an equal volume of phenol/chloroform and the BAC DNA precipitated with two volumes of ethanol.(e) After the final spin at 4,500 × g for 30 min at 4°C, the DNA pellet was air-dried and resuspended in 20 m l sterile ddH 2O. 2. PCR verification: multiple colonies with the correct antibiotic sensitivities were picked for the mini-BAC DNA preparations. The ORF deletions with Kan R replacements were confirmed by PCR using a HotStar DNA polymerase kit following a standard protocol. The target ORF should be absent in ORF deletion clones while the adjacent ORFs should remain intact as positive controls. 3. Maxi-BAC DNA preparations: the large-scale BAC DNA preparations using the NucleoBond Xtra Maxi Plasmid DNA purification kit (Clontech Laboratories, Inc., Palo Alto, CA) started with 500 ml of overnight cultures. The standard man-ufacturer’s protocol for BAC DNA purification was followed. The final DNA products were resuspended in 250 m l sterile ddH 2O and quantified by spectroscopy (see Note 9). 4. Hin dIII digestion profiling: PCR verified clones were selected for maxi-BAC DNA preparations. To confirm that no large VZV genomic DNA segment is deleted, Hin dIII digestion profiling was routinely carried out (see Note 10). Three micrograms of BAC DNA from maxi-preparations were digested with 20 U of Hin dIII in a 20-m l reaction at 37°C overnight. Hin dIII digestion patterns were compared by electrophoresis on ethidium bromide stained 0.5% agarose gels. As shown in Fig. 1, Hin dIII digestion patterns of each VZV ORF deletion clone were highly comparable with the parental wild-type VZV luc clone (see Note 11).The generation of VZV ORF deletion revertants is necessary to prove that the deleted ORF is responsible for any phenotype (usu-ally a growth defect) observed in analyses of the deletion mutants. The viral revertants should be able to fully restore the wild-type phenotype. As an example, generating the VZV ORFX deletion rescue virus is described to demonstrate the approach (see Fig. 2).1. VZV ORFX was amplified from wild-type VZV luc BAC DNAby PCR. Two unique restriction enzyme sites and two addi-tional 6-bp random sequences were added to the ends of the PCR product. A hi-fidelity PCR kit could be used in order to minimize unwanted mutations during PCRs (see Note 2).3.2. Generation of VZVORF DeletionRevertant BAC Clones82Zhang, Huang, and Zhu2. The ORFX gene was directionally cloned into pGEM-zeo to form pGEM-ORFX-zeo. The cloned ORFX was verified by sequencing analysis.3. ORFX-zeoR cassette was made by PCR using pGEM-ORFX-zeo as template (Fig. 2). The PCR product contained 40-bp homologies of flanking sequences of Kan R cassette, which was also used to generate the ORFX deletion mutant.4. The subsequent procedures are similar to producing the ORFX deletion mutant. Briefly, the linear ORFX-zeoR cas-sette was treated with DpnI and electroporated into compe-tent DY380 cells harboring VZV luc ORFX deletion BAC. Similarly, homologous recombination functions were tran-siently induced by switching the culture temperature from 32 to 42°C for 10–15 min when electroporation-competent cells were prepared. The recombinants were selected on LB agar plates containing zeocin. After verification, the ORFX dele-tion rescue BAC DNA was isolated from E. coli .Because of VZV’s highly cell-associated nature in cell culture,conventional virology techniques, including plaque purification and plaque assay, become troublesome. By developing andexploiting the new luciferase VZV BAC system, the resulting virus has a removable EGFP expression cassette and a built-in 3.3. Transfectionand Subsequent Virological Assayszeo R lox mcs mcskan lox zeo R ORFX lox lox E. ORFXR D. ORFXR-zeo B. C. ORFXD zeo R ORFX zeo ORFX ORFX Fig. 2. Generating an ORFX deletion rescue clone (ORFXR). (a ) To generate the ORFXR clone, ORFX was amplified by PCR from the wild-type VZV BAC DNA. The ORFX was directionally cloned into plasmid pGEM-lox-zeo to form pGEM-zeo-ORFX. (b ) Amplification of the ORFX-Zeo R cassette by PCR using a primer pair adding 40 bp homologies flanking ORFX. (c ) Such PCR product was transformed into DY380 carrying the VZV luc ORFXD BAC via electroporation. (d ) Homologous recombination between upstream and downstream homologies of ORFX replaced Kan R with the ORFX-Zeo R cassette, creating the ORFXR clone. (e ) Zeo R was removed while generating virus from BAC DNA by co-transfecting a Cre recombinase expressing plasmid.83An Efficient Protocol for VZV BAC-Based Mutagenesis luciferase reporter. In this protocol, an alternative biolumines-cence quantification approach has been provided to significantly increase the reproducibility of results. This approach has also been successfully used in monitoring VZV growth in vivo (10). 1. VZV BAC DNA from maxi-preparations was transfected into MeWo cells using the FuGene6 transfection kit according to the manufacturer’s standard protocol. 2. One and a half micrograms of BAC DNA and 6 m l of transfec-tion reagent were used for a single reaction in one well of 6-well tissue culture plates (see Note 12). 3. As an option, 0.5 m g of Cre expression plasmid was co-transfected with the VZV BAC DNA to remove the BAC sequence flanked by two loxP site from the viral genome (see Note 13). 4. In order to prevent the precipitation of BAC in solution, 1.5 m g BAC DNA were diluted in serum-free tissue culture medium, and the volume of DNA solution was adjusted to 50 m l (see Note 14). 5. The DNA solution was slowly added to the transfection reagent with gentle stirring using pipet tips. 6. Because of GFP expression from the BAC vector, VZV plaques were usually visually discernable using a fluorescent microscope within 3–5 days after transfection given deleted ORF is dispensable (see Note 15). If a VZV ORF is essential for viral replication, no plaque will be observed. 7. Since VZV is highly cell-associated in tissue culture, mutant VZV-infected MeWo cells were harvested and stored in liquid nitrogen for future studies.Recombinant viruses were titered by infectious focus assay. MeWo cells were seeded in 6-well tissue culture plates and inoculated with serial dilutions of VZV-infected MeWo cell suspensions. Plaques were counted by fluorescent microscopy 3 days after inoculation and viral titer was determined. 1. MeWo cells were infected with 100 PFU of infected MeWo cell suspensions in 6-well tissue culture plates. 2. After every 24-h interval, cell culture media was replaced with media containing 150 m g/ml d -luciferin.3. After incubation at 37°C for 10 min, the bioluminescent sig-nal was quantified and recorded using an IVIS ImagingSystem following the manufacturer’s instructions. 4. Fresh tissue culture medium was added to replace the luciferin-containing medium for further incubation at later time points.3.3.1. Transfection of BACDNA into MeWo Cells3.3.2. Titering by InfectiousFocus Assay3.3.3. Growth CurveAnalyses Based onBioluminescence Imaging(See Fig. 3 and Note 16)84Zhang, Huang, and Zhu5. Measurements from the same plate were repeated every day for 7 days.6. Bioluminescence signal data from each sample was quantified by manual designation of regions of interest and analyzed using Living Image analysis software (see Note17).1. The luciferase expression cassette, driven by an SV40 early pro-moter, was inserted between VZV ORF65 and ORF66. The cassette also contains a hygromycin B resistance gene (Hyg R ).2. Platinum Taq DNA polymerase can be used alternatively if a hi-fidelity PCR product is preferred.3. In order to achieve optimum results, the final concentration of the linear DNA cassette for the subsequent electroporation was adjusted to at least 100 ng/m l.4. The 42°C temperature shift is critical for the success of the homologous recombination. The temperature needs to be adjusted accurately to 42°C and remain constant. Too much recombination system activity is detrimental to E. coli and harm the integrity of BAC DNA. On the other hand, inade-quate induction of the recombination system in DY 380 leads to inefficient recombination. Ten to fifteen minutes might need to be adjusted carefully in order to achieve optimized efficiency of homologous recombination.5. E. coli DY380 strain needs to be cultured at 32°C all the time except when the recombination system is transiently activated and expressed by shifting the culture to 42°C.6. Beyond this point, every step needs to be carried out at a low temperature (0–4°C). All reagents, centrifuge rotor and glass-ware need to be prechilled.4. N otes Growth curve analysisVZVluc infectedMeWo cells / animal. a b c d Bioluminescenceimaging Image acquisition Fig. 3. Growth curve analyses based on bioluminescence imaging. (a ) Small animals/tissue culture can be infected with VZV luc . (b ) After administration of an enzyme substrate, luciferin, bioluminescence emitting from living animals/cultured cells can be detected and monitored by using a bioluminescence imaging system (a CCD camera mounted on top of a light-tight imaging dark chamber). (c ) Data can be stored in a connected PC and quantified by using region-of-interest analysis. (d ) Viral growth kinetics can be analyzed based on quantification of bioluminescence signals.85 An Efficient Protocol for VZV BAC-Based Mutagenesis7. Recombinants often have multiple antibiotic resistances. For instance, VZV ORFX/Kan clone will have Kan R, Cm R (from BAC vector), and Hyg R (from luciferase cassette). Screening for recombinants with more than one antibiotic is optional. However, the growth rate under such conditions could be much slower than selection under one antibiotic.8. If a clone also has Amp R, it should count as a false positive result.9. Due to the large size, handling BAC DNAs needs to avoid any harsh physical sheering force including vortexing or quickly passing through fine pipette tips. Freeze and thaw should also be avoided. BAC DNA solutions should always be stored at 4°C.10. Although it has been shown that VZVluc DNA is highly stablein E. coli (10) under the conditions described in this protocol, large undesirable deletions in the BAC clones were observed if homo l ogous recombination system in DY380 was over-induced.11. Since many large DNA fragments are generated by a Hin dIIIdigestion of the VZV genome, smaller genetic alterations, including replacement of an ORF by a Kan R cassette, would be difficult to recognize by this assay.12. The ratio of BAC DNA and FuGene6 reagent might need tobe adjusted to maximize transfection efficiency.13. The ORFX rescue clone was generated by introducing the wild-type ORFX back into the deletion viral genome along with a Zeo R cassette flanked by two loxP sites. By following this optional step in transfection, Zeo R will be removed from the genome by Cre-mediated recombination. The resulting virus will have a wild-type copy of ORFX restored in the same direction and loca-tion as the parental wild-type strain except a remaining loxP site(34 bp) in the 3¢ noncoding region of ORFX.14. Highly concentrated (greater than 250 m g/m l) BAC DNAsolutions are viscous and BAC DNA molecules easily precipi-tate out of solution when added to transfection reagent solu-tions. When such precipitation becomes visible, it is irreversible and the result of the transfection assays is often poor.Therefore, we predilute each BAC DNA in media before gen-tly mixing with the transfection reagent.15. Transfection efficiency was easy to monitor because of theresulting GFP expression from the BACs.16. Growth curve analyses were traditionally carried out by aplaque assay-based method.17. 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Cell-Type-Specific Differentiation andMolecular Profilesin Skin Transplantation: Implication ofMedical Approachfor Genetic Skin Diseases1.Introduction 前言In skin transplantation biology, multistage and multifocal damages occur in both grafted donor and perilesional host skin and need to be repaired properly for the engraftment and maintenance of characteristic skin architecture.在皮肤移植生物学中，多级和多焦损害发生在嫁接的捐助和高血压宿主皮肤和需要妥善修复植入和维护皮肤典型体系结构。
These local events are more unlikely to be regulated by the host immunity, because human skin transplantation has accomplished the donor skin engraftment onto the immunocompromised or immunosuppressive animals. Recent这些地方的事件是受宿主免疫，更可能的因为人体皮肤移植取得捐助者皮肤植入到免疫缺陷或免疫抑制剂动物。
From a dermatological viewpoint, we review the recent update of cell-type- and molecular-specific action associated with reconstitution of the grafted skin and also focus on the novel application of BM transplantation medicine in genetic skin diseases.从皮肤病学的角度来看，我们审查与移植的皮肤重建相关联的特定细胞类型和分子操作新的更新，也侧重于BM 移植医学在遗传皮肤疾病中应用。
中国生物医学文献英文全拼China's Biomedical Literature in Full English SpellingIntroductionBiomedical research plays a crucial role in advancing healthcare, improving medical treatments, and understanding diseases. As a global leader in scientific research, China has made significant contributions to the field of biomedicine. This article aims to explore the topic of Chinese biomedical literature and discuss the importance of using full English spellings in scientific publications.Benefits of English Spelling in Biomedical Literature1. Global Reach and CollaborationUsing full English spellings in biomedical literature allows researchers from around the world to access and understand the research findings. English is the most widely spoken language in scientific communities, and using it enables seamless collaboration among scientists from different countries. It helps overcome language barriers and fosters the exchange of knowledge and ideas.2. Standardization and ClarityAdopting full English spellings in biomedical literature promotes standardization and clarity in scientific communication. It ensures consistent terminology and facilitates accurate translations. Standardized English spellings enable researchers to clearly identify and interpret scientific terms, minimizing confusion and misunderstandings in scientific discourse.3. Visibility and ImpactEnglish is the dominant language in scientific publications, and papers published using full English spellings tend to reach a larger audience. This increased visibility can enhance the impact of Chinese biomedical research globally, attracting more attention and citations from scientists worldwide. It also improves the chances of collaborations, funding opportunities, and recognition for Chinese researchers.Challenges and Solutions1. Language BarrierLanguage proficiency can be a challenge for Chinese researchers when writing in English. To overcome this, universities and research institutions can provide additional support, such as English language training and scientific writing workshops. Collaborations with native English-speaking researchers can also help improve the quality of written English in scientific publications.2. Cultural DifferencesCultural nuances can affect the clarity and effectiveness of scientific communication in English. Chinese researchers should strive to understand and adapt to the cultural norms of scientific writing in English-speaking countries. This includes proper citation conventions, avoiding excessive jargon, and following the established structure and formatting guidelines of scientific papers.3. Translator Tools and DatabasesTo ensure accurate full English spelling of Chinese biomedical literature, the development of reliable translator tools specifically designed for scientific publications is essential. These tools can assist researchers in translating Chinese scientific terms into their appropriate English equivalents. Creating comprehensive databases of commonly used Chinese biomedical terms and their English translations can also be beneficial.Contribution of Chinese Biomedical LiteratureChinese biomedical literature has made significant contributions to the global scientific community. It has provided valuable insights into various areas of research, including pharmaceuticals, biomedical engineering, genetics, and disease prevention. Chinese researchers have published groundbreaking studies on diseases such as cancer, infectious diseases, and cardiovascular conditions, significantly advancing medical knowledge and treatment options.ConclusionThe use of full English spellings in Chinese biomedical literature is crucial for global visibility, collaboration, and impact. By overcoming language barriers and promoting standardized communication, Chinese researchers can enhance the reach and significance of their scientific contributions. Continued efforts in improving English language proficiency, cultural adaptation, and the development of reliable translator tools will further empower Chinese scientists in their pursuit of biomedical excellence.。
The future of biomedical materials生物医用材料的展望James M. Anderson詹姆斯·M·安德森Abstract摘要The purpose of this communication is to present the author’s perspectives on the future of biomedical materials that were presented at the Larry L. Hench Retirement Symposium held at Imperial College, London, in late September 2005.这次交流的目的是为了表达作者的观点，这个观点是在2005年9月后期在伦敦帝国学院举行的Larry L. Hench退休座谈会上被提出来的。
The author has taken a broad viewof the future of biomedical materials and has presented key ideas, concepts, and perspectives necessary for the future research and development of biomedical polymers and their future role as an enabling technology for the continuing progress of tissue engineering, regenerative medicine, prostheses, and medical devices.作者放眼生物医用材料的未来前景并介绍了主要观点、概念、将来实验所必须的透视图、生物医用材料的发展以及将来作为组织工程学连续发展、再生医药、假肢、医疗设备等的授权工艺。
中英文对照翻译Carotenoid Biosynthetic Pathway in the Citrus Genus: Number of Copies and Phylogenetic Diversity of SevenGeneThe first objective of this paper was to analyze the potential role of allelic variability of carotenoid biosynthetic genes in the interspecifi diversity in carotenoid composition of Citrus juices. The second objective was to determine the number of copies for each of these genes. Seven carotenoid biosynthetic genes were analyzed using restriction fragment length polymorphism (RFLP) and simple sequence repeats (SSR) markers. RFLP analyses were performed with the genomic DNA obtained from 25 Citrus genotypes using several restriction enzymes. cDNA fragments of Psy, Pds, Zds, Lcyb, Lcy-e, Hy-b, and Zep genes labeled with [R-32P]dCTP were used as probes. For SSR analyses, two primer pairs amplifying two SSR sequences identified from expressed sequence tags (ESTs) of Lcy-b and Hy-b genes were designed. The number of copies of the seven genes ranged from one for Lcy-b to three for Zds. The genetic diversity revealed by RFLP and SSR profiles was in agreement with the genetic diversity obtained from neutral molecμLar markers. Genetic interpretation of RFLP and SSR profiles of four genes (Psy1, Pds1, Lcy-b, and Lcy-e1) enabled us to make inferences on the phylogenetic origin of alleles for the major commercial citrus species. Moreover, the resμLts of our analyses suggest that the allelic diversity observed at the locus of both of lycopene cyclase genes, Lcy-b and Lcy-e1, is associated with interspecific diversity in carotenoid accumμLation in Citrus. The interspecific differences in carotenoid contents previously reported to be associated with other key steps catalyzed by PSY, HY-b, and ZEP were not linked to specific alleles at the corresponding loci.KEYWORDS: Citrus; carotenoids; biosynthetic genes; allelic variability; phylogeny INTRODUCTIONCarotenoids are pigments common to all photosynthetic organisms. In pigment-protein complexes, they act as light sensors for photosynthesis but also prevent photo-oxidation induced by too strong light intensities. In horticμLtural crops, they play a major role in fruit, root, or tuber coloration and in nutritional quality. Indeed some of these micronutrients are precursors of vitamin A, an essential component of human and animal diets. Carotenoids may also play a role in chronic disease prevention (such as certain cancers), probably due to their antioxidant properties. The carotenoid biosynthetic pathway is now well established. Carotenoids are synthesized in plastids by nuclear-encoded enzymes. The immediate precursor of carotenoids (and also of gibberellins, plastoquinone, chlorophylls,phylloquinones, and tocopherols) is geranylgeranyl diphosphate (GGPP). In light-grown plants, GGPP is mainly derivedcarotenoid, 15-cis-phytoene. Phytoene undergoes four desaturation reactions catalyzed by two enzymes, phytoene desaturase (PDS) and β-carotene desaturase (ZDS), which convert phytoene into the red-colored poly-cis-lycopene. Recently, Isaacson et al. and Park et al. isolated from tomato and Arabidopsis thaliana, respectively, the genes that encode the carotenoid isomerase (CRTISO) which, in turn, catalyzes the isomerization of poly-cis-carotenoids into all-trans-carotenoids. CRTISO acts on prolycopene to form all-trans lycopene, which undergoes cyclization reactions. Cyclization of lycopene is a branching point: one branch leads to β-carotene (β, β-carotene) and the other toα-carotene (β, ε-carotene). Lycopene β-cyclase (LCY-b) then converts lycopene intoβ-carotene in two steps, whereas the formation of α-carotene requires the action of two enzymes, lycopene ε- cyclase (LCY-e) and lycopene β-cyclase (LCY-b). α- carotene is converted into lutein by hydroxylations catalyzed by ε-carotene hydroxylase (HY-e) andβ-carotene hydroxylase (HY-b). Other xanthophylls are produced fromβ-carotene with hydroxylation reactions catalyzed by HY-b and epoxydation catalyzed by zeaxanthin epoxidase (ZEP). Most of the carotenoid biosynthetic genes have been cloned and sequenced in Citrus varieties . However, our knowledge of the complex regμLation of carotenoid biosynthesis in Citrus fruit is still limited. We need further information on the number of copies of these genes and on their allelic diversity in Citrus because these can influence carotenoid composition within the Citrus genus.Citrus fruit are among the richest sources of carotenoids. The fruit generally display a complex carotenoid structure, and 115 different carotenoids have been identified in Citrus fruit. The carotenoid richness of Citrus flesh depends on environmental conditions, particμLarly on growing conditions and on geographical origin . However the main factor influencing variability of caro tenoid quality in juice has been shown to be genetic diversity. Kato et al. showed that mandarin and orange juices accumμLated high levels of β-cryptoxanthin and violaxanthin, respectively, whereas mature lemon accumμLated extremely low levels of carotenoids. Goodner et al. demonstrated that mandarins, oranges, and their hybrids coμLd be clearly distinguished by their β-cryptoxanthin contents. Juices of red grapefruit contained two major carotenoids: lycopene and β-carotene. More recently, we conducted a broad study on the organization of the variability of carotenoid contents in different cμLtivated Citrus species in relation with the biosynthetic pathway . Qualitative analysis of presence or absence of the different compounds revealed three main clusters: (1) mandarins, sweet oranges, and sour oranges; (2) citrons, lemons, and limes; (3) pummelos and grapefruit. Our study also enabled identification of key steps in the diversification of the carotenoid profile. Synthesis of phytoene appeared as a limiting step for acid Citrus, while formation of β-carotene and R-carotene from lycopene were dramatically limited in cluster 3 (pummelos and grapefruit). Only varieties in cluster 1 were able to produce violaxanthin. In the same study , we concluded that there was a very strong correlation between the classification of Citrus species based on the presence or absence of carotenoids (below,this classification is also referred to as the organization of carotenoid diversity) and genetic diversity evaluated with biochemical or molecμLar markers such as isozymes or randomLy amplified polymorphic DNA (RAPD). We also concluded that, at the interspecific level, the organization of the diversity of carotenoid composition was linked to the global evolution process of cμLtivated Citrus rather than to more recent mutation events or human selection processes. Indeed, at interspecific level, a correlation between phenotypic variability and genetic diversity is common and is generally associated with generalized gametic is common and is generally associated with generalized gametic disequilibrium resμLting from the history of cμLtivated Citrus. Thus from numerical taxonomy based on morphological traits or from analysis of molecμLar markers , all authors agreed on the existence of three basic taxa (C. reticμLata, mandarins; C. medica, citrons; and C. maxima, pummelos) whose differentiation was the resμLt of allopatric evolution. All other cμLtivated Citr us species (C. sinensis, sweet oranges; C. aurantium, sour oranges; C. paradisi, grapefruit; and C. limon, lemons) resμLted from hybridization events within this basic pool except for C. aurantifolia, which may be a hybrid between C. medica and C. micrantha .Our previous resμLts and data on Citrus evolution lead us to propose the hypothesis that the allelic variability supporting the organization of carotenoid diversity at interspecific level preceded events that resμLted in the creation of secondary speci es. Such molecμLar variability may have two different effects: on the one hand, non-silent substitutions in coding region affect the specific activity of corresponding enzymes of the biosynthetic pathway, and on the other hand, variations in untranslated regions affect transcriptional or post-transcriptional mechanisms.There is no available data on the allelic diversity of Citrus genes of the carotenoid biosynthetic pathway. The objective of this paper was to test the hypothesis that allelic variability of these genes partially determines phenotypic variability at the interspecific level. For this purpose, we analyzed the RFLPs around seven genes of the biosynthetic pathway of carotenoids (Psy, Pds, Zds, Lcy-b, Lcy-e, Hy-b, Zep) and the polymorphism of two SSR sequences found in Lcy-b and Hy-b genes in a representative set of varieties of the Citrus genus already analyzed for carotenoid constitution. Our study aimed to answer the following questions: (a) are those genes mono- or mμLtilocus, (b) is the polymorphism revealed by RFLP and SSR markers in agreement with the general history of cμLtivated Citrus thus permitting inferences about the phylogenetic origin of genes of the secondary species, and (c) is this polymorphism associated with phenotypic (carotenoid compound) variations.RESΜLTS AND DISCUSSIONGlobal Diversity of the Genotype Sample Observed by RFLP Analysis. RFLP analyses were performed using probes defined from expressed sequences of seven major genes of the carotenoid biosynthetic pathway . One or two restriction enzymes were used for each gene. None of these enzymes cut the cDNA probe sequence except HindIII for the Lcy-e gene. Intronic sequences and restriction sites on genomic sequences werescreened with PCR amplification using genomic DNA as template and with digestion of PCR products. The resμLts indicated the absence of an intronic sequence for Psy and Lcy-b fragments. The absence of intron in these two fragments was checked by cloning and sequencing corresponding genomic sequences (data not shown). Conversely, we found introns in Pds, Zds, Hy-b, Zep, and Lcy-e genomic sequences corresponding to RFLP probes. EcoRV did not cut the genomic sequences of Pds, Zds, Hy-b, Zep, and Lcy-e. In the same way, no BamHI restriction site was found in the genomic sequences of Pds, Zds, and Hy-b. Data relative to the diversity observed for the different genes are presented in Table 4. A total of 58 fragments were identified, six of them being monomorphic (present in all individuals). In the limited sample of the three basic taxa, only eight bands out of 58 coμLd not be observed. In the basic taxa, the mean number of bands per genotype observed was 24.7, 24.7, and 17 for C. reticμLata, C. maxima, and C. medica, respectively. It varies from 28 (C. limettioides) to 36 (C. aurantium) for the secondary species. The mean number of RFLP bands per individual was lower for basic taxa than for the group of secondary species. This resμLt indicates that secondary species are much more heterozygous than the basic ones for these genes, which is logical if we assume that the secondary species arise from hybridizations between the three basic taxa. Moreover C. medica appears to be the least heterozygous taxon for RFLP around the genes of the carotenoid biosynthetic pathway, as already shown with isozymes, RAPD, and SSR markers.The two lemons were close to the acid Citrus cluster and the three sour oranges close to the mandarins/sweet oranges cluster. This organization of genetic diversity based on the RFLP profiles obtained with seven genes of the carotenoid pathway is very similar to that previously obtained with neutral molecμLar markers such as genomic SSR as well as the organization obtained with qualitative carotenoid compositions. All these resμLts suggest that the observed RFLP and SSR fragments are good phylogenetic markers. It seems consistent with our basic hypothesis that major differentiation in the genes involved in the carotenoid biosynthetic pathway preceded the creation of the secondary hybrid species and thus that the allelic structure of these hybrid species can be reconstructed from alleles observed in the three basic taxa.Gene by Gene Analysis: The Psy Gene. For the Psy probe combined with EcoRV or BamHI restriction enzymes, five bands were identified for the two enzymes, and two to three bands were observed for each genotype. One of these bands was present in all individuals. There was no restriction site in the probe sequence. These resμLts lead us to believe that Psy is present at two loci, one where no polymorphism was found with the restriction enzymes used, and one that displayed polymorphism. The number of different profiles observed was six and four with EcoRV and BamHI, respectively, for a total of 10 different profiles among the 25 individuals .Two Psy genes have also been found in tomato, tobacco, maize, and rice . Conversely, only one Psy gene has been found in Arabidopsis thaliana and in pepper (Capsicum annuum), which also accumμLates carotenoids in fruit. According to Bartley and Scolnik, Psy1 was expressed in tomato fruit chromoplasts, while Psy2 was specific to leaf tissue. In the same way, in Poaceae (maize, rice), Gallagher et al. found that Psy gene was duplicated and that Psy1 and notPsy2 transcripts in endosperm correlated with endosperm ca rotenoid accumμLation. These resμLts underline the role of gene duplication and the importance of tissue-specific phytoene synthase in the regμLation of carotenoid accumμLation.All the polymorphic bands were present in the sample of the basic taxon genomes. Assuming the hypothesis that all these bands describe the polymorphism at the same locus for the Psy gene, we can conclude that we found allelic differentiation between the three basic taxa with three alleles for C. reticμLata, four for C. maxima, and o ne for C. medica.The alleles observed for the basic taxa then enabled us to determine the genotypes of all the other species. The presumed genotypes for the Psy polymorphic locus are given in Table 7. Sweet oranges and grapefruit were heterozygous with one mandarin and one pummelo allele. Sour oranges were heterozygous; they shared the same mandarin allele with sweet oranges but had a different pummelo allele. Clementine was heterozygous with two mandarin alleles; one shared with sweet oranges and one with “Willow leaf” mandarin. “Meyer” lemon was heterozygous, with the mandarin allele also found in sweet oranges, and the citron allele. “Eureka”lemon was also heterozygous with the same pummelo allele as sour oranges and the citron allele. The other acid Citrus were homozygous for the citron allele.The Pds Gen. For the Pds probe combined with EcoRV, six different fragments were observed. One was common to all individuals. The number of fragments per individual was two or three. ResμLts for Pds led us to bel ieve that this gene is present at two loci, one where no polymorphism was found with EcoRV restriction, and one displaying polymorphism. Conversely, studies on Arabidopsis, tomato, maize, and rice showed that Pds was a single copy gene. However, a previous study on Citrus suggests that Pds is present as a low-copy gene family in the Citrus genome, which is in agreement with our findings.The Zds Gene. The Zds profiles were complex. Nine and five fragments were observed with EcoRV and BamHI restriction, respectively. For both enzymes, one fragment was common to all individuals. The number of fragments per individual ranged from two to six for EcoRV and three to five for BamHI. There was no restriction site in the probe sequence. It can be assumed that several copies (at least three) of the Zds gene are present in the Citrus genome with polymorphism for at least two of them. In Arabidopsis, maize, and rice, like Pds, Zds was a single-copy gene .In these conditions and in the absence of analysis of controlled progenies, we are unable to conduct genetic analysis of profiles. However it appears that some bands differentiated the basic taxa: one for mandarins, one for pummelos, and one for citrons with EcoRV restriction and one for pummelos and one for citrons with BamHI restriction. Two bands out of the nine obtained with EcoRV were not observed in the samples of basic taxa. One was rare and only observed in “Rangpur” lime. The other was found in sour oranges, “V olkamer” lemon,and “Palestine sweet” lime suggesting a common ancestor for these three genotypes.This is in agreement with the assumption of Nicolosi et al. that “V olkamer” lemon resμLts from a complex hybrid combination with C. aurantium as one parent. It will benecessary to extend the analysis of the basic taxa to conclude whether these specific bands are present in the diversity of these taxa or resμLt from mutations after the formation of the secondary species.The Lcy-b Gene with RFLP Analysis.After restriction with EcoRV and hybridization with the Lcy-b probe, we obtained simple profiles with a total of four fragments. One to two fragments were observed for each individual, and seven profiles were differentiated among the 25 genotypes. These resμLts provide evidence that Lcy-b is present at a single locus in the haploid Citrus genome. Two lycopene β-cyclases encoded by two genes have been identified in tomato. The B gene encoded a novel type of lycopene β-cyclase whose sequence was similar to capsanthin-capsorubin synthase. The B gene expressed at a high level in βmutants was responsible for strong accumμLation ofβ-carotene in fruit, while in wild-type tomatoes, B was expressed at a low level.The Lcy-b Gene with SSR Analysis. Four bands were detected at locus 1210 (Lcy-b gene). One or two bands were detected per variety confirming that this gene is mono locus. Six different profiles were observed among the 25 genotypes. As with RFLP analysis, no intrataxon molecμLar polymorphism was found within C. Paradisi, C. Sinensis, and C. Aurantium.Taken together, the information obtained from RFLP and SSR analyses enabled us to identify a complete differentiation among the three basic taxon samples. Each of these taxons displayed two alleles for the analyzed sample. An additional allele was identified for “Mexican” lime. The profiles for all secondary species can be reconstructed from these alleles. Deduced genetic structure is given in. Sweet oranges and clementine were heterozygous with one mandarin and one pummelo allele. Sour oranges were also heterozygous sharing the same mandarin allele as sweet oranges but with another pummelo allele. Grapefruit were heterozygous with two pummelo alleles. All the acid secondary species were heterozygous, having one allele from citrons and the other one from mandarins ex cept for “Mexican” lime, which had a specific allele.柑桔属类胡萝卜素生物合成途径中七个基因拷贝数目及遗传多样性的分析摘要：本文的首要目标是分析类胡萝卜素生物合成相关等位基因在发生变异柑橘属类胡萝卜素组分种间差异的潜在作用；第二个目标是确定这些基因的拷贝数。
