Marker Insertion Research Articles

Marker-based technique for visualizing radiolucent implant components in radiographic imaging.

Radiography is the predominant imaging modality used for the in-vivo analysis of orthopaedic implants. A major disadvantage of radiography is that the articulating joint components that are composed of radiolucent polyethylene cannot be directly visualized. Current strategies attempt to circumvent this limitation by estimating component positions and simplifying the joint system, however, these approaches lead to a number of associated errors. Thus, this study provides a method to enable the visualization of the polyethylene component of total knee replacements in radiographic images. This was achieved through the repeatable insertion of markers and accompanying registration process, which were evaluated in this study for reproducibility and accuracy. An insertion guide was developed to insert tantalum beads into polyethylene tibial surface liners. The bead-inserted liners were micro-CT scanned to obtain 3D surface geometries. An in-vivo mimicking phantom RSA experiment was then used to test the 3D to 2D registration process. The guide positioned the beads consistently to ±0.21 mm. The 3D to 2D registration demonstrated a repeatability of -0.014 ± 0.008 mm. Registration of different bead-inserted tibial liners to the phantom revealed an average error of 0.026 ± 0.047 mm for this visualization method. This visualization approach provides greatly improved registration and inter-component measurements than current alternative strategies. This process is suitable for a number of other joints and would greatly benefit procedures that analyze component interactions and implant performance over time. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2017-2022, 2017.

Does body mass index or subcutaneous adipose tissue thickness affect interfraction prostate motion in patients receiving radical prostate radiotherapy?

AbstractAimIt is unclear whether body mass index (BMI) is a useful measurement for examining prostate motion. Patient’s subcutaneous adipose tissue thickness (SAT) and weight has been shown to correlate with prostate shifts in the left/right direction. We sought to analyse the relationship between BMI and interfraction prostate movement in order to determine planning target volume (PTV) margins based on patient BMI.Materials and methodsIn all, 38 prostate cancer patients with three implanted gold fiducial markers in their prostate were recruited. Height, mass and SAT were measured, and the extent of interfraction prostate movement in the left/right, superior/inferior and anterior/posterior directions was recorded during each daily fiducial marker-based image-guided radiotherapy treatment. Mean corrective shift in each direction for each patient, along with BMI values, were calculated.ResultsThe median BMI value was 28·4 kg/m2 (range 21·4–44·7). Pearson’s product-moment correlation analysis showed no significant relationship between BMI, mass or SAT and the extent of prostate movement in any direction. Linear regression analysis also showed no relationship between any of the patient variables and the extent of prostate movement in any direction (BMI: R2=0·006 (ρ=0·65), 0·002 (ρ=0·80) and 0·001 (ρ=0·86); mass: R2=0·001 (ρ=0·87), 0·010 (ρ=0·54) and 0·000 (ρ=0·99); SAT: R2=0·012 (ρ=0·51), 0·013 (ρ=0·50) and 0·047 (ρ=0·19) for shifts in the X, Y and Z axis, respectively). Patients were grouped according to BMI, as BMI<30 (n=25, 65·8%) and BMI≥30 (n=13, 34·2%). A two-tailed t-test showed no significant difference between the mean prostate shifts for the two groups in any direction (ρ=0·320, 0·839 and 0·325 for shifts in the X, Y and Z axis, respectively).FindingsBMI is not a useful parameter for determining individualised PTV margins. Gold fiducial marker insertion should be used as standard to improve treatment accuracy.

Target margins in radiotherapy of prostate cancer.

We reviewed the literature on the use of margins in radiotherapy of patients with prostate cancer, focusing on different options for image guidance (IG) and technical issues. The search in PubMed database was limited to include studies that involved external beam radiotherapy of the intact prostate. Post-prostatectomy studies, brachytherapy and particle therapy were excluded. Each article was characterized according to the IG strategy used: positioning on external marks using room lasers, bone anatomy and soft tissue match, usage of fiducial markers, electromagnetic tracking and adapted delivery. A lack of uniformity in margin selection among institutions was evident from the review. In general, introduction of pre- and in-treatment IG was associated with smaller planning target volume (PTV) margins, but there was a lack of definitive experimental/clinical studies providing robust information on selection of exact PTV values. In addition, there is a lack of comparative research regarding the cost-benefit ratio of the different strategies: insertion of fiducial markers or electromagnetic transponders facilitates prostate gland localization but at a price of invasive procedure; frequent pre-treatment imaging increases patient in-room time, dose and labour; online plan adaptation should improve radiation delivery accuracy but requires fast and precise computation. Finally, optimal protocols for quality assurance procedures need to be established.

PWE-088 UK Pilot Study of EUS Guided Fiducial Marker Placement for Image Guided Radiotherapy in Oesophageal Cancer: Initial Results of Feasibility and Radiotherapy Outcomes

Introduction Fiducial markers (FM) help in radiotherapy planning (RT) by improving target delineation and optimise image guided radiotherapy (IGRT) during RT delivery. It helps in reducing interfraction RT setup uncertainty and improving the accuracy of treatment delivery. So far there are no studies in the UK that examined this technique with use of FM in Oesophageal cancers. We are hereby reporting the findings of our UK pilot study of FM guided 3 D Conformal RT and IGRT. Aim To evaluate the clinical outcomes of endoscopic ultrasound guided insertion of fiducial markers to demarcate oesophageal cancer for IGRT for Lancashire and South Cumbria cancer network (LSCCN) patients. Methods Institutional research and ethics committee approval was obtained. Our charity Rosemere Cancer Foundation funded this Prospective non-randomised cohort study. Cook EchoTip Ultra EUS Needles 22-FTM preloaded with 4 gold fiducial markers were used. Each marker measured 10 mm in length and 0.25μ in diameter. OlympusTM linear EUS was used to initially stage the tumour, followed by insertion of FM at the inferior and superior margins of the tumour. All patients with Oesophageal Squamous cancers at LSCCN from January 2015 to January 2016 considered for chemoradiotherapy were assessed for FM assisted IGRT. Contraindications include non-traversable tumours and patients with Oesophageal stent in situ. Results In total, 13 patients had 47 FM inserted under EUS guidance. Immediate CXR visualisation of FM was seen in 43/49 (88%). EUS guided FM insertion was feasible in all patients, no adverse events reported. Only 11 patients were eventually fit to undergo radiotherapy, of which data was available on 8 patients who received Radical 3 D conformal RT with 50 Gy. Markers were visualised in 21/27 (75%) on CT planning and during IGRT at treatment delivery using cone-beam CTs. Migration of the FM was seen with 2 markers, close to the actual lesion (in 2 patients). FM derived gross tumour length (GTV) on planning CT was similar to endoscopic tumour length (7.4 cm vs 8 cm). Mean GTV volume was 40.09 cubic cm (range: 18.57–80.1). Mean GTV length was 7.4 cm (range: 3.75–11.3). Mean PTV volume was 252.07 cubic cm (range: 135.4–357.2). Mean V20 for Lung was 19.16% (range: 13.1–28.37). FM assisted significantly in RT planning for tumour delineation. Conclusion FM for oesophageal cancer is technically feasible, safe, significantly improves target delineation for radiotherapy with excellent visibility for IGRT and acceptable stability (7.4% migration rate). Our completed data comparing FM based IGRT with conventional IGRT will help further clarifying its routine use in RT for patients with squamous cell oesophageal cancers. Disclosure of Interest None Declared

Plant active LTR retrotransposons: a review

Long terminal repeat (LTR) retrotransposons are mobile DNA sequences that ubiquitously exist in eukaryotic genomes. They replicate themselves in the genome by copy-paste mechanism with RNA as medium. In higher plants, many active LTR retrotransposons have been applied to analyze molecular marker technology, genetic tagging, insertion mutation and gene function. Here, we systematically review the characteristics of plant active LTR retrotransposons, including their structures, copy numbers and distributions. We further analyzed the gag (group-specific antigen) and pol (polymerase) sequence features of different plants active LTR retrotransposons and the distribution patterns of the cis-acting elements in LTR regions. The results show that autonomous active LTR retrotransposons must contain LTR regions and code Gag, Pr, Int, Rt, Rh proteins. Both LTR regions are highly homologous with each other and contain many cis-regulatory elements; RVT and RNase_H1_RT domain are essential for Rt and Rh protein respectively. These results provide the basis for subsequent identification of plant active LTR retrotransposons and their functional analysis.

A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

Material-flow behavior during friction-stir welding of 6082-T6 aluminum alloy

Material-flow behavior during friction-stir welding of 6082-T6 aluminum alloy has been visualized by marker insert technique. Four stacked layers were evolved in welding nugget zone (WNZ) in the transverse section. The material-flow behavior in vertical direction was detected by observing the distribution of Cu foil fragments and Al-Cu intermetallic compounds. The downward and upward flows encounter each other at the advancing side in the material depositing process, changing the morphology of WNZ. A conceptual balanced-flow model and a plastic material-flow model were used to describe the material-flow behavior, and the origin of the downward material flow in the advancing side was discussed. The excess-material flow derived by the welding tool due to the existence of the tilt angle is crucial to the weld formation.

Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus

Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95–100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.

Strain and texture in friction extrusion of aluminum wire

Friction extrusion is a solid-state process that can produce high quality, fully consolidated wire or rod directly from metal chips, powder or billet. However, little is understood regarding the variation in material flow or extrusion strain with changes in processing parameters. Extrusion strain level may be of great import in determining whether or not the charge is fully consolidated. In order to explore the material deformation behavior during this process, flow visualization experiments were conducted using AA6061 billets with AA2195 as a marker insert. Variations in material flow during a single extrusion were documented and correlated with changes in grain size, which has previously been correlated with extrusion temperature. Marker shape was used to make an approximation of imposed strain during the extrusion as a function of relative extrusion temperature. Also, tests using various extrusion forces and die rotation speeds were conducted. The influence of extrusion parameters on deformation evolution was elucidated and discussed. Grain orientation analysis conducted using electron backscatter diffraction showed a fully recrystallized microstructure with weak texture indicating that recrystallization was likely a static process occurring after passage of the wire through the die. Key findings include: (1) longitudinal strain is solely a function of overall reduction (2) in plane shear strain decreases with increasing extrusion temperature, and (3) with increasing extrusion temperature, friction extrusion becomes similar to normal extrusion.

A novel endoscopic marker for radiological localization and image-guided radiotherapy in esophageal and gastric cancers (with video)

Radiotherapy is an accepted modality in the treatment of esophageal cancers and is currently being evaluated in conjunction with chemotherapy for the neoadjuvant treatment of gastric cancers. Our aim was to assess whether a novel endoscopically inserted marker can be used to improve radiological assessment of the primary cancer and allow for image-guided radiotherapy. A phase II feasibility study was conducted at a tertiary-care center. Twenty-six consecutive adult patients with esophagogastric cancers underwent endoscopic marking of the tumor margins with a novel radiopaque marker (mixture of lipiodol and n-butyl 2-cyanoacrylate). The main outcome measure was the successful insertion of the marker based on a combination of radiological, endoscopic, and histological assessment. A total of 92 markers were inserted in 26 patients. Twenty-two (88%) had follow-up imaging to assess the 81 markers inserted, 79 of which (97.5%) were visible. There were no postprocedural adverse events noted in our cohort. Radiological assessment of tumor size improved such that it was in line with the endoscopic evaluation after marker placement in 18 of 21 patients (85.7%) who had appropriate follow-up radiology imaging. Ten patients (38.5%) from our cohort underwent image-guided radiotherapy (IGRT) by using the endoscopically inserted markers. Within the limitations of our small pilot study, endoscopic placement of our novel marker was successful in the majority of our cohort without significant adverse events. Marker placement resulted in improved radiological localization in the majority of our cohort and allowed for IGRT. (Australian New Zealand Clinical Trials Registry: ACTRN12613000239763.).

Off-axis twist extrusion for uniform processing of round bars

The present paper introduces a twist extrusion (TE) process capable of processing of round bars with uniform deformation and reports physical, analytical, and numerical modeling of the process. It is shown that the ability to treat round bars can be achieved by design of special off-axis TE dies in which the axis of the twist surface is displaced from the central axis of the bar being processed. Physical modeling conducted in the current study with plasticine demonstrates the feasibility of off-axis TE. A marker insert technique employed in the physical model reveals that tool-controlled flow (ideal helical flow) of the material is dominant in the process. Analytical model developed in the present study explains why using off-axis TE dies leads to uniform deformation and how this deformation uniformity depends on the die geometry. The main conclusions made upon analytical modeling are confirmed with complement finite element simulations. The simulations also show that the main deformation mode in off-axis TE is simple shear at the intersection planes between the twist and the straight channels of the die.

Fiducial Marker Insertion for Image-guided Radiotherapy for Prostate Cancer: What is the Infection Rate and can Targeted Antibiotic Prophylaxis Reduce this?

Fiducial Marker Insertion for Image-guided Radiotherapy for Prostate Cancer: What is the Infection Rate and can Targeted Antibiotic Prophylaxis Reduce this?

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a ‘targeting’ vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Ultrasonography-guided surgical clip placement for tumor localization in patients undergoing neoadjuvant chemotherapy for breast cancer.

PurposeWe investigated the feasibility of using surgical clips as markers for tumor localization and their effect on the imaging evaluation of treatment responses after neoadjuvant chemotherapy (NAC).MethodsA total of 16 breast cancers confirmed by needle biopsy in 15 patients were included in this study from October 2012 to June 2014. Under ultrasonography (US)-guidance, the surgical clips were placed prior to NAC. Additional mammography, breast US, and breast magnetic resonance examinations were performed within 10 days before surgery. The time period from marker insertion to operation date was documented. Images acquired via the three modalities were evalu-ated for the following parameters: location of clip, clip migration (>1 cm), the presence of complications from clip placement, and the effect of clips on the assessment of treatment.ResultsThe mean time period was 128.6±34.4 days (median, 132.0 days) from the date of clip insertion to the date of surgery. The mean number of inserted clips was 2.3±0.7 (median, 2.0). Clip migration was not visualized by imaging in any patient, and there were no complications reported. Surgical clips did not negatively affect the assessment of treatment responses to NAC.ConclusionSurgical clips may replace commercial tissue markers for tumor localization in breast cancer patients undergoing NAC without migration. Surgical clips are well tolerated and safe for the patient, easily visualized on imaging, do not interfere with treatment response, and are cost-effective.

Robotically Guided Prostatic Fiducial Marker Insertion Under Direct MRI Guidance: A Proof of Concept in a Live Canine Subject

Materials/Methods: We calculated VHEE treatment plans for a lung cancer case using the EGSnrc/DOSxyznrc code implemented in an inhouse MATLAB based GUI developed by our group. We evaluated the use of beam energies: 80 MeV, 100 MeV, and 120 MeV; number of equidistant beams: 16 or 32; and beamlets size: 3 mm, 5 mm or 7 mm. Inverseplanning optimization was performed in a research version of a commercially available treatment planning software using identical objective functions and constraints for all VHEE plans. We used the corresponding clinical 6MV photon VMAT, calculated in an Eclipse Treatment Planning System, for comparison purposes. The prescription dose (Dp) was 5400 cGy to 95% of the 99 cm planning target volume (PTV). We calculated the conformity index defined as the ratio of the 50% (CI50) and 100% (CI100) isodose volume to the PTV volume. Results: PTV coverage was matched and indexes CI50 and CI100 were equivalent for all the VHEE plans. Average dose to organs at risk (OARs) was equivalent for the 100 MeV and 120 MeV plans. The 80 MeV plan showed up to 8% higher average doses than the 100 MeV plan. Based in these results we selected 100MeV as optimum energy. The study to determine the optimum number of beams (16 or 32) showed a mean difference in average dose of 2.4% (0%-7.7%) between the two plans. For simplicity and due to the small differences found we chose 16 beams as the optimum number of beams. The study of the beamlet size showed that a 3 mm beamlet size systematically reduces the dose to all the OARs. Based on these results we selected the 100 MeV-16 beams-3 mm beamlet size as our best VHEE plan to compare it against VMAT. Average Doses were lower for the VHEE plan. The larger differences were found in the low dose region (<25%Dp). Heart (125% lower dose) and Trachea (177% lower dose) were the structures that benefited the most from using VHEE. Average dose to the body was 11% higher for VMAT. VHEE plan was more conformal than VMAT (CI50 index was 14% higher for the VMAT). Conclusions: Using 100 MeV-16 beams-3 mm beamlet size of VHEE for the planning of a lung cancer case provided the highest dose sparing of OARs, improving the actually delivered VMAT plan. These parameters could be used for other targets with similar size, shape, and location. For larger targets a larger beamlet size such as 5mm might be used without significantly increasing the dose. Author Disclosure: B.A. Palma: None. M. Bazalova: None. H. Bjorn: A. Employee; RaySearch Americas. E. Hynning: A. Employee; RaySearch Americas. B. Qu: None. P.G. Maxim: E. Research Grant; Varian, RaySearch. F. Honoraria; Varian. B.W. Loo: E. Research Grant; Varian, RaySearch. F. Honoraria; Varian.

Trafficking of bluetongue virus visualized by recovery of tetracysteine-tagged virion particles.

Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses. Live-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

Novel Technique for Hepatic Fiducial Marker Placement for Stereotactic Body Radiation Therapy

To report experience with fiducial marker insertion and describe an advantageous, novel technique for fiducial placement in the liver for stereotactic body radiation therapy with respiratory tracking. We implanted 1444 fiducials (single: 834; linked: 610) in 328 patients with 424 hepatic lesions. Two methods of implantation were compared: the standard method (631 single fiducials) performed on 153 patients from May 2007 to May 2010, and the cube method (813 fiducials: 610 linked/203 single) applied to 175 patients from April 2010 to March 2013. The standard method involved implanting a single marker at a time. The novel technique entailed implanting 2 pairs of linked markers when possible in a way to occupy the perpendicular edges of a cube containing the tumor inside. Mean duration of the cube method was shorter than the standard method (46 vs 61 minutes; P<.0001). Median numbers of skin and subcapsular entries were significantly smaller with the cube method (2 vs 4, P<.0001, and 2 vs 4, P<.0001, respectively). The rate of overall complications (total, major, and minor) was significantly lower in the cube method group compared with the standard method group (5.7% vs 13.7%; P=.013). Major complications occurred while using single markers only. The success rate was 98.9% for the cube method and 99.3% for the standard method. We propose a new technique of hepatic fiducial implantation that makes use of linked fiducials and involves fewer skin entries and shorter time of implantation. The technique is less complication-prone and is migration-resistant.

AglB, catalyzing the oligosaccharyl transferase step of the archaeal N‐glycosylation process, is essential in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius

Sulfolobus acidocaldarius, a thermo-acidophilic crenarchaeon which grows optimally at 76°C and pH 3, exhibits an astonishing high number of N-glycans linked to the surface (S-) layer proteins. The S-layer proteins as well as other surface-exposed proteins are modified via N-glycosylation, in which the oligosaccharyl transferase AglB catalyzes the final step of the transfer of the glycan tree to the nascent protein. In this study, we demonstrated that AglB is essential for the viability of S. acidocaldarius. Different deletion approaches, that is, markerless in-frame deletion as well as a marker insertion were unsuccessful to create an aglB deletion mutant. Only the integration of a second aglB gene copy allowed the successful deletion of the original aglB.

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

ABSTRACTToxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.

Fine Mapping of the Maize Cross‐Incompatibility Locus Gametophytic Factor 1 (ga1) Using a Homogeneous Population

ABSTRACTPhenotyping of a mapping population is usually the bottleneck that limits the size of the mapping population and mapping resolution. A homogeneous population mapping approach was used for mapping maize (Zea mays L.) gametophytic factor 1 (ga1) that could completely eliminate phenotyping during the mapping process. The strong allele of maize ga1 (Ga1‐S), from popcorn inbred line SDGa25, showed a 100% cross‐incompatibility with the majority of Chinese dent and flint maize. A homogeneous mapping population was developed by crossing an (SDGa25/Jing66) F1 male back to an SDGa25 female. During the pollination process, pollen grains of ga1 were completely excluded from fertilization, and a homogeneous BC1F1 (SDGa25//SDGa25/Jing66) population was created in which only the Ga1‐S/Ga1‐S genotype existed, making phenotyping unnecessary. A total of 2245 individuals of this population were genotyped with SD9 and SD12 markers and 20 recombinants were identified. The Ga1‐S locus was quickly delineated to a 100 Kb region between markers dCS1 and insertion deletion ID7 at position 9,491,422 and 9,591,946 bp based on the B73 RefGen_v2 sequence. By marker‐assisted selection, Ga1‐S was introgressed into parental lines of an elite white waxy maize hybrid by six generations of backcrossing and one generation of selfing. The homozygous Ga1‐S/Ga1‐S hybrid showed full cross‐incompatibility to ga1 maize. The Ga1‐S allele could be used as a biological reproductive barrier in reducing cross‐pollination between different types of maize such as waxy and non‐waxy, genetically modified (GM) and non‐GM maize.