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The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.

The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

The bottleneck in elucidating gene function through high-throughput gain-of-function genome screening is the limited availability of comprehensive libraries for gene overexpression. Lentiviral vectors are the most versatile and widely used vehicles for gene expression in mammalian cells. Lentiviral supernatant libraries for genome screening are commonly generated in the HEK293T cell line, yet very little is known about the effect of introduced sequences on the produced viral titer, which we have shown to be gene dependent. We have generated an arrayed lentiviral vector library for the expression of 17,030 human proteins by using the GATEWAY® cloning system to transfer ORFs from the Mammalian Gene Collection into an EF1alpha promoter-dependent lentiviral expression vector. This promoter was chosen instead of the more potent and widely used CMV promoter, because it is less prone to silencing and provides more stable long term expression. The arrayed lentiviral clones were used to generate viral supernatant by packaging in the HEK293T cell line. The efficiency of transfection and virus production was estimated by measuring the fluorescence of IRES driven GFP, co-expressed with the ORFs. More than 90% of cloned ORFs produced sufficient virus for downstream screening applications. We identified genes which consistently produced very high or very low viral titer. Supernatants from select clones that were either high or low virus producers were tested on a range of cell lines. Some of the low virus producers, including two previously uncharacterized proteins were cytotoxic to HEK293T cells. The library we have constructed presents a powerful resource for high-throughput gain-of-function screening of the human genome and drug-target discovery. Identification of human genes that affect lentivirus production may lead to improved technology for gene expression using lentiviral vectors.

The protein tyrosine phosphatase receptor J, PTPRJ, is a tumor suppressor gene that has been implicated in a range of cancers, including breast cancer, yet little is known about its role in normal breast physiology or in mammary gland tumorigenesis. In this paper we show that PTPRJ mRNA is expressed in normal breast tissue and reduced in corresponding tumors. Meta-analysis revealed that the gene encoding PTPRJ is frequently lost in breast tumors and that low expression of the transcript associated with poorer overall survival at 20 years. Immunohistochemistry of PTPRJ protein in normal human breast tissue revealed a distinctive apical localisation in the luminal cells of alveoli and ducts. Qualitative analysis of a cohort of invasive ductal carcinomas revealed retention of normal apical PTPRJ localization where tubule formation was maintained but that tumors mostly exhibited diffuse cytoplasmic staining, indicating that dysregulation of localisation associated with loss of tissue architecture in tumorigenesis. The murine ortholog, Ptprj, exhibited a similar localisation in normal mammary gland, and was differentially regulated throughout lactational development, and in an in vitro model of mammary epithelial differentiation. Furthermore, ectopic expression of human PTPRJ in HC11 murine mammary epithelial cells inhibited dome formation. These data indicate that PTPRJ may regulate differentiation of normal mammary epithelia and that dysregulation of protein localisation may be associated with tumorigenesis.

NEW Queensland Firebirds coach Vicki Wilson has retained the majority of last season's squad despite many being on the brink of retirement. Wilson has kept defensive trio Peta Stephens, pictured, Kelly-Ann MacKenzie and Melanie McKenzie, as well as former national squad player Lauren Nourse. Wilson, who said the outlook on former national squad shooter Carla Dziwoki also looked promising, will meet this weekend with former Firebird goal attack Belinda Dever. MacKenzie said she and Stephens wanted to be part of the Firebirds program with Wilson at the helm. \"At the end of last season I wasn't coming back but Vicki is on another level,\" she said. Wilson will now look at promising youngsters and those representing Queensland at this week's national titles in Canberra, where the Maroons play for fifth position today after beating the ACT 69-33. THREE Queensland Commonwealth Games hopefuls leave next week for a final international hit-out before selection trials in December but Maroon teammate Hayley Wright has been a late injury withdrawal. Wright last week retore the thigh muscle that hampered her world championship selection campaign. She is on a modified training program for at least a month but should be ready for the December 9- 10 selection trials in Canberra. The six-woman team for the Massilia Gymnastics Cup in France and the Australia-Italy challenge in Pavia, Italy will be led by 2008 Olympic squad members Alyce Arrowsmith, pictured, and Naomi Russell and Jasmine Webb. National junior champion Arrowsmith is hoping to put behind her a long-term ankle injury. AIS athlete Victoria Williams has replaced Wright for the tour, joined by teammates Ashleigh Brennan and Melody Hernandez.

DUAL Olympian Troy Elder has failed in a one-to-one bid to convince Australian hockey coach Barry Dancer to reselect him for the Commonwealth Games team. But Dancer has given Elder, pictured, his blessing to play club hockey again in The Netherlands and will strongly consider him for Australia's World Cup campaign in Germany next year. Elder remains disappointed at his year-long suspension imposed after he failed to attend the Azlan Shah Cup in Malaysia in May. The event clashed with the Dutch club final series. \"I never did anything illegal,\" he said. \"It was only one week out of the whole year that I was unavailable for Australia.\" Elder has not lost hope that Dancer may reconsider his position but Dancer was adamant Elder was out until next March. Elder's Dutch club side won the grand final. UNBEATEN Queensland Oaks winner White Thunder has the best form of any horse in Australia but surprisingly is rated only third favourite in tonight's Australian Oaks (2570m) at Moonee Valley. White Thunder is chasing her 14th straight win but in UniTAB's fixed odds betting yesterday she was rated $3.75 behind Victorian Oaks winner Innocent Eyes ($3) and New Zealand filly Molly Darling ($3.20), who has been placed in the Victoria, NSW and NZ Oaks. Shoalhaven (NSW) trainer Chris Robinson, pictured, said he could not agree with White Thunder's assestment, although the Victorian factor of champion reinsman Gavin Lang on polemarker Innocent Eyes could influence price assessors. \"We will be going forward from barrier five and expect Molly Darling to try to hold the lead,\" Robinson said.

EASTERN Suburbs punished Taringa's inability to finish the game when it fought back from a 3-1 deficit to snatch a 3-3 draw in the Brisbane Premier League match at Jack Speare Park on Saturday. Easts began its comeback when Michael Thompson capitalised on some sloppy defending to score. Thompson also netted the equaliser. Brisbane City continued its gloomy start to the season with a 3-2 away loss to Toowoomba Raiders. Mitchelton posted its second win of the season with a 4-1 thrashing of the University of Queensland. Palm Beach moved to joint first place after its 2-1 home win over rivals Rochedale Rovers, while Brisbane Strikers drew 1-1 with Pine Rivers, who equalised with a goal by Josh Rose, pictured, at Perry Park. Souths United maintained its perfect record with a 3-1 win over Brisbane Wolves. FOUR years ago, Chris McCormack, pictured, shed tears of pain and emotion to win the Australian ironman triathlon on debut. Yesterday, he cried again as he raced to a record-equalling fourth successive title in the 20th anniversary race in Forster. The win came 13 years after a teenage McCormack had headed to the NSW coastal town to watch his Finnish idol Pauli Kiuru win the second of his four consecutive Australian ironman crowns -- a feat McCormack matched yesterday. McCormack conquered the arduous 3.8km swim, 180km cycle and 42.2km run in 8hr 25min 45sec a day shy of his 32nd birthday. His feat was replicated less than an hour later by Canadian Lisa Bentley who matched Australian Louise Bonham's feats of 1987, 1988, 1989 and 1992 in winning a fourth women's crown in 9:13.22.

ANTHONY Mundine, pictured, will shun hometown Sydney and seek to win the world super-middleweight title in Brisbane or Melbourne, with his manager claiming NSW boxing officials are out to destroy his career. Mundine's manager Khoder Nasser said the former world champion loved NSW but wanted nothing to do with the state's boxing authorities after they refused to sanction Wednesday night's fight against Darmel Castillo because of fears for the lowly ranked fighter's safety. \"The NSW Minister for Sport (Sandra Nori) should get rid of the lot of them or ban boxing in NSW,\" Nasser said. \"You've got these seriously pathetic people trying to destroy the career of a great Australian sportsman.\" Mundine delivered the 18th KO of his career in the fourth round to put Castillo on the canvas in front of a crowd of about 1500 at the Gold Coast Convention Centre. SISTERS Anna and Kerry Meares will face each other for the first time since Anna's Olympic win and Kerry's injury comeback at next week's national track titles in Adelaide. The Meares sisters are members of one of the strongest Queensland teams, which leaves for Adelaide today and includes three Olympic/Paralympic gold medallists and three world champions. Anna, pictured, the Olympic, world and national 500m time-trial champion, and Kerry, the Commonwealth Games and former national 500m time-trial titleholder, will each contest the time trial, the sprint and keirin. Olympic road champion Sara Carrigan will race the individual pursuit in her first assault on open track. Athens Olympic weightlifter Deborah Lovely will compete in her first national cycling titles.

TOP Queensland coach Graeme Brimblecombe, pictured inset, is coming home after accepting a position as Tennis Queensland director of player development. Brimblecombe, who has coached at the NSW Institute of Sport for two years, will manage a regional Queensland Academy of Sport program and oversee the state's player development system from November. TQ chief executive Steve Ayles said the recruitment of Brimblecombe added to optimism in the sport in Queensland in the week after confirmation that a state centre at Tennyson would be built. Brimblecombe has been working at the NSWIS with 16-year-old Queenslander Joel Lindner, pictured, winner of an ITF event in Hungary in May. He is likely to receive an AIS scholarship. DRIVER Charlie Cini expects the favourite She's A Lefty to go one better than her dam Lefty to win the $75,000 Triad 2yo Fillies final at Albion Park tomorrow night. \"She's drawn to win from barrier three,\" he said yesterday. \"I plan to lead. My main danger is the Graeme Bowyer-trained Bonnies The Best who is off the back row.\" Cini drives the Perfect Art filly for his wife Cristina Monte, who trains at Jacobs Well. She's A Lefty is the second foal from Lefty, who ran second in the 1997 Triad before winning the Qbred Breeders in 1:59.2. She's A Lefty has emulated her dam, winning last month's Breeders at the Gold Coast in 1:57.7. Top Kiwi reinsman Anthony Butt, pictured, who won the 2001 Brisbane Interdominion trot on Take A Moment, has taken the drive on Armbro Included, replacing local Joel Rees.