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Archive for December, 2006

An Important Notice

Posted by tumicrobiology on December 20, 2006

Dear all, 

We, in collaboration with our partner HealthandBiology, are going to launch our next site very soon.

There will be more features and more gifts to you!

A gate way to the Nepali journals and much more. We are negotiating with Nepali journal publishers.

You will be fascinated with what we are bringing, if we succeed.

Pray for our success.

Thanking you,

Gaffer, NMDF

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HIV Patients Have Increased Risk Of Pneumonia, Death Following Surgery

Posted by tumicrobiology on December 20, 2006

Science Daily HIV-infected patients undergoing surgical procedures may be more likely to develop pneumonia after surgery and to die within 12 months than those without HIV, according to a report in the December issue of Archives of Surgery, one of the JAMA/Archives journals. In addition, HIV patients with a preoperative viral load (number of copies of the virus in the blood) greater than 30,000 per milliliter appear to have increased risk of surgical complications.

Since the development of medication regimens known as highly active antiretroviral therapy (HAART), HIV has become a chronic, manageable condition, according to background information in the article. “Consequently, many HIV-infected patients elect to undergo surgical procedures to correct physical ailments that would not have been treated previously, and undergo operative interventions in lieu of medical therapies for certain conditions,” the authors write.

Michael A. Horberg, M.D., M.A.S., and colleagues at Kaiser Permanente Medical Care Program–Northern California, Oakland, studied surgical outcomes in 332 HIV-infected patients who underwent a variety of procedures (including abdominal, orthopedic and heart surgeries) between 1997 and 2002. For comparison, the researchers selected a group of 332 patients who did not have HIV but were the same age and sex and had a similar procedure at around the same time and at the same location as one of the HIV-infected patients. The investigators then used health plan databases to obtain clinical information about the HIV patients’ disease and to track whether any of the patients had complications after surgery or died within 12 months.

The surgical procedures analyzed included abdominal or pelvic procedures (80.8 percent), cardiac or breast procedures (8.4 percent) and orthopedic procedures (10.8 percent). Most complications–including infections and delayed wound healing–occurred equally frequently in patients with and without HIV. No difference between the two groups was found in the length of hospital stay, number of complications or need for additional procedures to treat complications. However, more HIV patients developed pneumonia (eight or 2.4 percent vs. one or .3 percent) and more died within 12 months (10 or 3 percent vs. two or .6 percent). “The causes of death varied” in HIV patients, the authors write. “While none of the causes appeared to be a direct consequence of the operation, two deaths were within 30 days of the operation.”

The researchers also examined risk factors for complications and death among HIV patients, including CD4 cell count response, a measure of the state of the immune system. The lower the CD4 count, the more likely a patient with HIV/AIDS is to develop secondary infections or illnesses. Those with a CD4 count of less than 50 cells per cubic millimeter of blood had more complications than those with higher CD4 counts. In addition, viral loads–measured as the number of copies of the virus in a milliliter of blood–of more than 30,000 were associated with a higher complication rate. Whether the patients were taking antiretroviral therapy did not appear to be related to their risk of developing complications. “Our results indicate that a higher HIV viral load seems to be a greater predictor of surgically related complications than either the CD4 cell count or the presence or absence of HAART use,” the authors write.

“Patients with HIV are living longer and regaining a substantial amount of immune function,” they conclude. “Many HIV-infected patients will require surgical attention because of a variety of disorders. In many cases, HIV serostatus [whether a person is infected with HIV or not] should not be a criterion when determining the need for surgery if patients have adequate viral control.”

Note: This story has been adapted from a news release issued by JAMA and Archives Journals.

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Bacterial infection ID method created

Posted by tumicrobiology on December 20, 2006

UPI— US scientists say they’ve developed a method of identifying specific sites of localized bacterial infections in living animals.
Bradley Smith of the University of Notre Dame and colleagues previously developed fluorescent molecular probes containing zinc that could be used to discriminate between common pathogenic bacteria, such as E. coli and Staphylococcus aureus, and mammalian cells.
In the new study, the scientists used the probes to pinpoint the sites of staph infections in laboratory mice. The scientists say physicians might have difficulty distinguishing localized bacterial infections from sites of sterile inflammation.
“Bacterial imaging is an emerging technology that has many health and environmental applications,” the researchers said. “For example, there is an obvious need to develop highly sensitive assays that can detect very small numbers of pathogenic bacterial cells in food, drinking water or biomedical samples. In other situations, the goal is to study, in vivo, the temporal and spatial distribution of bacteria in live animals.”
The study is described in a report scheduled for the Jan. 10 issue of the Journal of the American Chemical Society.
Copyright 2006 by United Press International. All Rights Reserved.

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Scientists Find Potential Weapon Against Tuberculosis Infection

Posted by tumicrobiology on December 14, 2006

The discovery of a unique copper-repressing protein in the bacterium that causes tuberculosis in humans may pave the way toward new strategies for halting tuberculosis infection.

Scientists have known that when macrophages – the host’s immune cells – swallow an invading bacterium, they dump excessive amounts of copper onto the invader in an effort to kill it. While all cells need copper to function, too much of the metal ion causes cell death.

“But the invaders fight back with their own defense,” says Adel Talaat, a microbiologist at the University of Wisconsin-Madison School of Veterinary Medicine. “They block the excess copper.”

In a paper published in the January 2007 issue of Nature Chemical Biology, Talaat and colleagues from Texas A&M University and University of Saskatchewan in Saskatoon, Canada describe a unique protein repressor that they have identified as the mechanism used by invading bacterium cells to fight off the host’s copper attack.

Prior to the discovery of this repressor protein, scientists did not know exactly how invading bacterium protected themselves from copper ions used by the body as a defense against infection.

“With this discovery, we can now pursue ways to deactivate the repressor protein,” says Talaat. “Our goal is to disable the tuberculosis bacterium from fighting back against the host body’s defense mechanisms, so that we can stop tuberculosis.”
Source: University Of Wisconsin-Madison

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Study: How shigella causes dysentery

Posted by tumicrobiology on December 14, 2006

French scientists say they believe they’ve discovered how shigella bacteria survive in the gut to be able to cause dysentery.

Laurence Arbibe and colleagues at the Pasteur Institute in Paris found a protein produced by shigella is injected into host cells and blocks production of immune signals required for preventing the infection.

Shigella affects millions of people worldwide, killing hundreds of thousands annually.

The researchers studied Shigella flexneri infection of human colon cells to understand the bacterial factors required to initiate disease. Shigella bacteria are known to inject up to 20 proteins into intestinal cells for the purpose of promoting infection and for dampening immune responses.

They found one of the injected proteins, OpsF, could prevent gut cells from switching on genes involved in immune responses. As a consequence, Shigella flexneri avoids being killed by their host’s immune cells and is able to spread throughout the gut.

The scientists say their study highlights the precision with which pathogens such as shigella can dramatically alter host cells. It also suggests blocking OspF may provide a target for treating bacterial dysentery.

The study is reported in the January issue of the journal Nature Immunology.

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Three studies focus on malaria parasite

Posted by tumicrobiology on December 14, 2006

The US and British scientists have produced three studies that independently characterize the genetic diversity of the parasite that causes malaria.

Two studies focused on Plasmodium falciparum, the most deadly of the Plasmodium species known to cause human malaria, while the other study compares it with the related Plasmodium reichenowi, which infects chimpanzees.

Overall, the scientists say the data constitute a valuable resource that should improve understanding of drug resistance in malaria and identify candidate targets for vaccines.

Dyann Wirth and colleagues at the Harvard School of Public Health produced a genome-wide map of diversity in P. falciparum, including full sequencing of 16 new and geographically diverse strains and targeted sequencing of 54 other worldwide isolates.

Xin-zhuan Su, Philip Awadalla and colleagues at the U.S. National Institutes of Health focus on sequencing genomic regions coding for proteins within 4 P. falciparum isolates.

In the third study, Manolis Dermitzakis, Matthew Berriman and colleagues at the Wellcome Trust Sanger Institute in Hinxton, England, provide the first sequence of the P. reichenowi strain, as well as the sequence of two P. falciparum strains.

All three papers appear in the January issue of Nature Genetics.

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Flu Shot Effective Against Drifted Influenza, Nasal Spray Vaccine Less So

Posted by tumicrobiology on December 14, 2006

During a year in which the circulating strains of influenza showed genetic differences from the strains in vaccines, the traditional killed-virus flu shot was found to be effective in preventing influenza in healthy adults. The live attenuated-virus nasal spray vaccine also prevented illnesses but was less effective.

Both outcomes were determined by laboratory confirmation of flu infection, a University of Michigan study found.

Earlier studies had suggested that the nasal spray, sold by MedImmune as FluMist, might offer better protection against drifted viruses that had genetically changed between vaccine formulation and annual influenza activity. The nasal spray, which is based on a live but weakened virus, was 86 percent protective in one study conducted in children during a major drift year. However, FluMist had not previously been studied head-to-head against the shot in adults with laboratory confirmation.

The killed-virus flu shot is usually billed as 70 to 90 percent effective against circulating strains that are well matched to vaccine strains. During the 2004-2005 flu season, a University of Michigan team found that the killed-virus flu shot was 75 percent effective against a moderately-drifted type A virus and two types of B virus. The standard formulation of both the flu shot and the nasal spray vaccine includes two types of A influenza and one B, but in the 2004-05 season, there were two B strains circulating and one type A.

“On the other hand, FluMist was 48 percent effective. These results may only apply to the 2004-05 flu season,” said Dr. Arnold S. Monto, professor of epidemiology. “In other years the results may be different. We need a more specific understanding of which viral changes matter and which don’t. There are many things about vaccine protectiveness that we still don’t completely understand.”

“It may be that the difference in effectiveness between the shot and the spray can be attributed to poorer protection against type B infections in participants given FluMist,” Monto said, but that’s not clear from this study.

The live attenuated vaccine marketed as FluMist was developed at the University of Michigan by Hunein “John” Massaab, professor emeritus of epidemiology. MedImmune produces it under a license with the University.

Monto’s research team is conducting a randomized, double-blind, placebo-controlled three-year trial with National Institutes of Health funding in which the two vaccines are being compared head-to-head and against a placebo. Prior to the 2004-05 flu season, they vaccinated 1,247 people aged 18 to 46 in four Michigan communities.

A source of confusion about vaccine effectiveness against drifted viruses may stem from study design. Many vaccine evaluation studies rely on a clinical diagnosis of “influenza-like illness,” or measures of the immune response to infection in the blood. For the present study, Monto and lead author Suzanne Ohmit, assistant research scientist in epidemiology, took throat swab specimens from participants experiencing flu symptoms and analyzed them, using virus isolation and PCR techniques, to determine if influenza virus was causing the illness.

Monto and Ohmit suspect that the adult participants in their study in 2004-05 had enough prior experience with influenza that the live attenuated virus may have failed to infect their nasal passages and initiate an immune response as it is intended to do. However, both still believe that the nasal spray is very effective in children and may be more effective than the killed vaccine in the young.

“FluMist works very well in children with naive immune systems,” Ohmit said. The FDA approval of FluMist limits its use to people between age 5 and 49 years.

In another paper in the same edition of the New England Journal of Medicine, FluMist was administered to school children and the children were followed to see if they and their families had been protected against flu. The study’s authors conclude that vaccinating children had some protective effect on the children and their families, confirming a notion of “herd immunity” first tested by Arnold Monto in a 1970 influenza vaccine study.

Without having children in their study, Monto and Ohmit can’t tell whether the two vaccines would have conferred different protection in 2004-05 in that age group. However, data from a study conducted in 2004-05 in children aged 6 to 59 months by MedImmune, suggested that FluMist offered significantly greater protection than the flu shot. A MedImmune study discussed in October at a Toronto conference also found that FluMist was more successful at initiating antibody response than the traditional shot in young children.

Monto and Ohmit will continue to follow study participants vaccinated in Fall 2005 , without vaccinating them this year, to see whether the shot or the spray offer long-term protection.

The paper “Prevention of Antigenically Drifted Influenza by Inactivated and Live Attenuated Vaccines,” appears in the Dec. 14 issue of the New England Journal of Medicine.
Source: University of Michigan

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Snottites, Other Biofilms Hasten Cave Formation

Posted by tumicrobiology on December 14, 2006

Biofilms, which are complex layered communities of sulfur-consuming microbes, increase the rate of cave formation, but may also shed light on other biofilms, including those that grow on teeth and those that corrode steel ships hulls, according to a team of geologists
Cave biofilms are simpler than the microbes that occur in soils where there can be hundreds of thousands of species,” says Dr. Jennifer L. Macalady, assistant professor of geosciences, Penn State. “Some cave biofilms have very few species, 10 to 20. The more complex ones have 100s or 1,000s.”

The researchers investigated the Frasassi cave system located north of Rome and south of Venice in Italy. These limestone caves are like New Mexico’s Carlsbad Caverns and Lechuguilla

Cave, but in those caves, sulfur entered the caves from oil and gas reserves, while in Italy, the sulfur source is a thick gypsum layer below. Having sulfur in the environment allows these biofilms to grow.

Most limestone caves form when rainwater and runoff permeate the caves from above. Water and carbon dioxide mix to form carbonic acid, a very weak acid, that erodes the limestone cave walls. In sulfidic caves, water enters the caves from below, carrying hydrogen sulfide. Microbes in the biofilms use the sulfur for energy and produce sulfuric acid, a very strong acid.

“One type of biofilm, called a snottite because of its appearance, has a pH of zero or one,” says Daniel S. Jones, graduate student in geosciences. “This is very, very acidic.”

Carbonic acid cave systems lose about a third of an inch of wall every thousand years, while sulfuric acid cave systems lose about two and a third inches or six times as much in the same time. The researchers are interested in the make-up of the biofilms and how they cycle sulfur.

Biofilms are made up of thin layers of microbe species that can be very different. All require water, but some biofilms live in the pools, lakes and streams in caves and others live on the damp walls. The layers against the rock surface use oxygen and hydrogen sulfide for energy and produce sulfuric acid. The layer on the outside does as well, but, because middle layers exist, there is an opportunity for microbes that find oxygen poisonous to thrive. These middle layers may convert the sulfuric acid to hydrogen sulfide, creating a complete sulfur cycle in a few microns.

In dental biofilms, the microbes on the teeth are the ones that produce the acids that cause cavities, while the ones on the top create the right conditions for the acid-producing microbes to survive. Cave biofilm layers also fulfill different niches in their very tiny environment.

“Stream biofilms are responsible for the majority of sulfide disappearance in streams,” Jones told attendees today (Dec. 11) at the fall meeting of the American Geophysical Meeting in San Francisco.

Because the cave biofilms are relatively simple, it will be easier to connect the various microbe species to the geochemistry involved. While this work is not yet complete, the researchers are working on the problem. Dr. Greg K. Druschel, assistant professor of geology, University of Vermont, used microelectrode voltammetry to try to determine exactly which biofilm layers produce acids. The levels of hydrogen sulfide and sulfuric acid change, depending on which layer is tested.

“There is also a question about where these microbes originate,” says Macalady. “We do not know if they are always in rocks or if they are transported from somewhere else to establish themselves.”

No matter the answer, the biofilms probably begin growing in tiny cracks in the rock and eventually create some of the largest cave systems in the world. Cave biofilms are also the bottom of the food chain for cave ecosystems. They provide food for a variety of spiders, flat worms, pill bugs and amphipods — shrimp like crustaceans — that form a blind and pigmentless community.

“The only other place we find sulfur-based ecosystems is near the deep sea vents on the ocean floor,” says Jones.

Understanding how cave biofilms dissolve calcium carbonate may help us to understand the communities around ocean floor vents, but it may also, eventually, lead to understanding how biofilms dissolve calcium phosphate on teeth and the steel hulls of ships.

Macalady, Jones and Druschel worked with two undergraduate students: Daniel Eastman, University of Vermont, and Lindsey Albertson, Brown University. The National Science Foundation and NASA Astrobiology institute supported this research.

Source: Penn State

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Male Circumcision Reduces HIV Risk, Study Stopped Early

Posted by tumicrobiology on December 14, 2006

A University of Illinois at Chicago study has been stopped early due to preliminary results indicating that medical circumcision of men reduces their risk of acquiring HIV during heterosexual intercourse by 53 percent.

The study’s independent Data Safety and Monitoring Board met Dec. 12 to review the interim data. Based on the board’s review, the National Institutes of Health halted the trial and recommended that all men enrolled in the study who remain uncircumcised be offered circumcision.

“Circumcision is now a proven, effective prevention strategy to reduce HIV infections in men,” said Robert Bailey, professor of epidemiology in the UIC School of Public Health and principal investigator of the study.

The clinical trial, funded by the National Institute of Allergy and Infectious Diseases and the Canadian Institute of Health Research, enrolled 2,784 HIV negative, uncircumcised men between 18 and 24 years old in Kisumu, Kenya.

Half the men were randomly assigned to circumcision, half remained uncircumcised. All men enrolled in the study received free HIV testing and counseling, medical care, tests and treatment for sexually transmitted infections, condoms and behavioral risk counseling for 24 months.

Study results show that 22 of the 1,393 circumcised men in the study contracted HIV, compared to 47 of the 1,391 uncircumcised men. In other words, circumcised men had 53 percent fewer HIV infections than uncircumcised men.

Until now, public health organizations have not supported circumcision as a method of HIV prevention due to a lack of randomized controlled trials.

“With these findings, the evidence is now available for donor and normative agencies, like WHO and UNAIDS, to actively promote circumcision in a safe context and along with other HIV prevention strategies,” Bailey said.

“Circumcision cannot be a stand-alone intervention. It has to be integrated with all the other things that we do to prevent new HIV infections, such as treating sexual transmitted diseases and providing condoms and behavioral counseling,” Bailey said. “We can’t expect to just cut off a foreskin and have the guy go on his merry way without additional tools to fight against getting infected.”

Opponents of circumcision have speculated that circumcised men may feel they are not at risk of contracting HIV and may be more likely to engage in risky behavior. The Kenya study suggests that circumcision did not increase risky behavior among circumcised or uncircumcised men, according to Bailey.

“Both uncircumcised and circumcised men are reducing their sexual risk behavior,” he said, “which indicates that our counseling is doing some good.”

The study also evaluated the safety of circumcision in a community health clinic with specially trained practitioners. There were no severe or lasting complications from circumcision. However, 1.7 percent of surgeries resulted in mild complications, such as bleeding or infection.

Bailey said that promoting circumcision in Africa must be done in conjunction with proper technical training and medical tools, equipment and supplies necessary to perform large numbers of circumcisions safely.

“Already, there are large numbers of boys and young men who are seeking circumcision in areas of Africa where men are not traditionally circumcised,” he said. “The danger is that unqualified practitioners will fill a niche by providing circumcision, but with much higher complication rates.”

An estimated 30 million people in Africa are infected with HIV/AIDS and more than 90 percent of HIV infections in adults result from heterosexual intercourse. In Kisumu, the third-largest city in Kenya, an estimated 26 percent of uncircumcised men are HIV infected by age 25.

“This study will likely not have a large impact on the incidence of HIV/AIDS in the United States or Europe where heterosexual transmission of HIV is low compared with areas like sub-Saharan Africa and parts of Asia,” Bailey said. “However, there are other proven health benefits of circumcision, including better hygiene, fewer urinary tract infections, and less risk of cervical cancer in the partners of circumcised men.”

The armamentarium of HIV prevention strategies is very small, according to Bailey. The only other strategy proven effective is the use of antiretroviral drugs to reduce transmission from mother to child.

If a significant proportion of men in a population get circumcised, it will have an enormous impact on preventing HIV infection in men, as well as reducing infections in women, Bailey said.

Co-investigators of the study include Stephen Moses and Ian Maclean at the University of Manitoba, Jekoniah Ndinya-Achola at the University of Nairobi, Corette Parker at Research Triangle International, Kawango Agot at UNIM Project, John Krieger at University of Washington, and Richard Campbell at UIC.

During the past two decades, more than 40 observational epidemiological studies and one previous clinical trial have reported an association between male circumcision and a reduced risk of HIV infection.

On Dec. 12, the NIH stopped another clinical trial of male circumcision undertaken by investigators in Uganda and at Johns Hopkins University, after the study’s Data Safety Monitoring Board reviewed the preliminary results and found a protective effect similar to that found in Bailey’s study.

Source: University of Illinois at Chicago
Date: December 13, 2006

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Natural Radioactivity Could Provide Microbes In The Deep Biosphere With Vitality

Posted by tumicrobiology on December 6, 2006

Source: Max-Planck-Gesellschaft

An international team of researchers from the USA and Germany has published an explanation for life in the deep biosphere in the journal Science. Using the latest technologies from biogeochemistry, molecular biology and microbiology, the scientists collected a wide range of samples from the bottom of the sea. After intensive analysis, Bo B. Jørgensen and Steven D´Hondt have now published a model with which they explain that microorganisms might survive due to the natural radioactivity deep under the sea floor (Science, 10th November 2006).

It is estimated today that between 10 and 50 percent of all the biomass on the Earth is found deep below ground. Researchers working with Steven D´Hondt from the University of Rhode Island, USA, and Bo B. Jørgensen from the Max Planck Institute for Marine Microbiology in Bremen have confirmed this in the course of the Ocean Drilling Program. On a voyage on the research ship “Joides Resolution” they found life up to 400 meters below the sea floor. Tests revealed that the drilled cores contained living microorganisms; contamination was ruled out. In the upper layers of sediment, the researchers counted up to 100 million unicellular organisms per millilitre; deeper, in the 35 million year old sediments on the Earth’s crust, they still found one million microorganisms. This is a puzzle for scientists: only the upper layers of these deposits are in contact with the water – so where does the energy to provide life in the depths of the sediment come from?

Taking as a basis the energy sources in the deposits that are available to the cells in the form of organic carbon compounds, it is possible to calculate that the cells could only divide every thousand years. This extremely long period for reproduction cannot be reconciled with current understanding of living cells.

Jørgensen and D’Hondt are now proposing, on the basis of their data, a process which could represent an alternative source of energy for life deep under large sections of the Pacific – natural radioactivity. Water is broken down by radioactive radiation, which arises during the decomposition of naturally occurring potassium, thorium and uranium isotopes. This process (radiolysis) creates hydrogen and oxygen. Estimates of the energy balance show that this process can supply sufficient energy for the microorganisms. This would make life forms in the Deep Biosphere independent of the processes on the Earth’s surface. The authors point out that an exotic habitat like this could also have developed on other planets, far away from any suns.

In December 2006 the researchers will be taking the drilling ship “RV Roger Revelle” to the South Pacific. There, far away from the continental shelves, the quantity of carbon compounds which could serve the microorganisms as a basis for life is very low. This makes the researchers all the more curious about the sediment samples from the bottom of the sea.

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Scientists spot unknown gene interaction

Posted by tumicrobiology on December 6, 2006

Italian genetic researchers say they’ve made a discovery that might provide an important tool for controlling and treating breast cancer.

Scientists at Milan’s National Tumor Institute discovered proteins produced by the genes HER-2 and FHIT interact in a way that encourages tumor growth, the Italian news agency ANSA reported Wednesday. The researchers determined HER-2 prevents FHIT from blocking cell proliferation. Without FHIT’s action, cells can multiply quickly and end up producing a tumor.

“The analysis carried out for our research shows how the activity of HER-2 leads to the degradation of FHIT,” Sylvie Menard, head of experimental oncology at INT, told ANSA.

Medical researchers previously determined breast tumor development was often accompanied by an overproduction of proteins by the HER-2 gene. It is also known that in 70 percent of breast cancer cases, FHIT has stopped working for some reason.

Menard told ANSA it should be possible to find pharmaceuticals to prevent FHIT degradation, thereby slowing the development of tumors.

The research appears in the current issue of the journal of the American Academy of the Sciences.

Copyright 2006 by United Press International. All Rights Reserved.

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Method for detection and identification of multiple chromosomal integration sites in transgenic animals created with lentivirus

Posted by tumicrobiology on December 4, 2006

Elizabeth C. Bryda, Michael Pearson, Yuksel Agca, and Beth A. Bauer

BioTechniques® December 2006
Volume 41, Number 6: pp 715-719

Abstract
Transgene delivery systems, particularly those involving retroviruses, often result in the integration of multiple copies of the transgene throughout the host genome. Since site-specific silencing of trangenes can occur, it becomes important to identify the number and chromosomal location of the multiple copies of the transgenes in order to correlate inheritance of the transgene at a particular chromosomal site with a specific and robust phenotype. Using a technique that combines restriction endonuclease digest and several rounds of PCR amplification followed by nucleotide sequencing, it is possible to identify multiple chromosomal integration sites in transgenic founder animals. By designing genotyping assays to detect each individual integration site in the offspring of these founders, the inheritance of transgenes integrated at specific chromosomal locations can be followed efficiently as the transgenes randomly segregate in subsequent generations. Phenotypic characteristics can then be correlated with inheritance of a transgene integrated at a particular chromosomal location to allow rational selection of breeding animals in order to establish the transgenic line.

INTRODUCTION

Production of genetically engineered animals by pronuclear injection or retroviral delivery systems has been a successful strategy for generating animal models to better understand the functionality of genes. In transgenic animals created by embryo microinjection, the site of integration of the transgene within the genome is a random event. Thus, when multiple embryos have been injected or infected with the same DNA, the integration site will be different in each founder animal. When the integration events have occurred at the one-cell stage, they should exhibit germline transmission with the potential to be inherited by the founder’s offspring. If the integration occurs at a later stage, the resulting mosaic founder may or may not exhibit germline transmission of the transgene. In the case of pronuclear injection, there is typically one insertion site, although multiple transgene copies are often found in a tandem array at that integration site (1).

Lentivirus transgenesis is becoming an increasingly attractive alternative to pronuclear injection because it is more efficient in terms of successful transgene incorporation into the host genome, less invasive to the embryo, and technically less demanding to perform (2). Lentiviral delivery systems have been used successfully to generate transgenic mice, rats, pigs, and cattle (2–7). The disadvantage of lentivirus is that there are often multiple integration events with random transgene insertions on several chromosomes.

Independent of the method of transgene delivery, the insertion site can have profound effects on transgene expression. This can lead to phenotypic effects in the transgenic animal that are not due to the transgene per se, but are a consequence of the integration site, a phenomenon referred to as position effect (8). It is critical to correlate phenotype with genotype, particularly in animals created via lentivirus transgenesis, since not all copies of the transgene may be contributing equally to the phenotype.

Determining transgene integration sites is challenging. A number of PCR-based methods, often referred to as chromosome walking techniques, have been developed to isolate DNA fragments adjacent to known sequences, including inverse PCR (9), ligation-mediated PCR (LMPCR) (10), randomly primed PCR (RP-PCR) (11,12), and T-linker PCR (13). The method described in this paper incorporates several elements of these techniques in a unique way that allows the capture of DNA fragments containing the chromosomal region flanking the transgene. Our method enables quick and inexpensive determination of multiple independent transgene integration sites in founder animals and their offspring. Here, we demonstrate that this method is useful for identifying and monitoring multiple transgene integration sites in transgenic animals created using lentivirus.

MATERIALS AND METHODS

Animals

Lewis rat lines carrying an enhanced green fluorescent protein (EGFP) transgene were created by using the EGFP DNA construct and experimental protocol described by Lois et al. (2). Transgene positive founder animals were identified using an EGFP PCR assay (14). To assess GFP expression, tail biopsies were examined for fluorescence under a Nikon SMZ1500 UV dissecting scope (Nikon Instruments, Melville, NY, USA). Founders (F0) were bred to wild-type Lewis rats obtained from Harlan Sprague Dawley (Indianapolis, IN, USA) to generate the N1. N1 animals were genotyped using integration site-specific PCR assays. Selected N1 animals were mated to wild-type Lewis rats to generate the N2. N2 animals were genotyped using integration site-specific genotyping assays, and GFP expression was confirmed. Several of these lines (F455.5, F456.9, F458.7, F463.1, and F463.5) demonstrated stable transmission of the integration site-specific transgene coupled with robust GFP expression and were donated to the Rat Research and Resource Center (RRRC) at the University of Missouri. All other rat lines and mouse strains used are available either through the RRRC (Web site (external)) for rats or the University of Missouri/Harlan Mutant Mouse Regional Resource Center (MMMRC; Web site (external)) for mice.

Preparation of DNA

DNA was isolated from tail biopsies using the DNeasy® Tissue kit (Qiagen, Valencia, CA, USA). Restriction endonuclease digestion was performed with PstI and HhaI (Figure 1, step 1). These enzymes were chosen because they created 3′ overhangs and their recognition sites were not present within the transgene sequence. Three micrograms genomic DNA were digested with 20 U enzyme in a total reaction volume of 30 μL as recommended by the manufacturer. Reactions were incubated for 2 h for partial digestion.

PCR Amplification and Linker Ligation

Three transgene-specific nested primers were designed to both the 5′ and 3′ regions of the transgene using PrimerQuest available from Integrated DNA Technologies (IDT; Coralville, IA, USA). Primer 1 was farthest from the junction between the known transgene sequence and the unknown integration site, whereas primer 3 was closest (Figure 1). All gene-specific primers were designed to have an optimum melting temperature (Tm) of 60°C, optimum primer length of 24 bp, and an optimum % GC content of 50. Y-linker and primer sequences for the Y-linker and their relationships are provided in Figure 2. Y-linker A (Figure 2) contains a terminal inverted T at the 3′ end to inhibit extension by DNA polymerases. The Y-linker was prepared by combining equal volumes of 8 μM Y-linker A and 8 μM Y-linker E, incubating at 95°C for 5 min, and then letting the reaction cool on the benchtop. The sequences of the genespecific primers used for the lentivirus-generated rat lines are available upon request or are listed under the genotyping assay for the specific lines at Web site (external). Primers and linkers were synthesized by IDT.

All PCRs were performed in a 25-μL volume containing 2.5 μL 10× FastStart Taq with 20 mM MgCl2 (Roche Diagnostics, Indianapolis, IN, USA), 0.2 mM each dNTP, 1.6 μM each primer, and 1.25 U FastStart Taq buffer. In the first PCR, 1 μL restriction digest from above and transgenespecific primer 1 were used, and a single round of PCR was performed with the following thermal cycling conditions: 94°C for 10 min, 60°C for 1 min, and 72°C for 10 min (Figure 1, step 2). Following amplification, a 10-μL reaction containing 7 μL this PCR, 1 μM Y-linker, 400 U T4 DNA Ligase (New England BioLabs, Ipswich, MA, USA), and 1 μL 10× T4 DNA Ligase buffer supplied with the enzyme was incubated at 16°C for 16 h (Figure 1, step 3). Transgene-specific primer 2, Y-primer D, and 1 μL ligase reaction were used in the second PCR with the following cycling conditions: 94°C for 5 min, 20 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1.5 min, and one cycle of 72°C for 10 min (Figure 1, step 4). The same cycling conditions were used in the third PCR. This final reaction utilized transgene-specific primer 3, Y-primer G, and 1 μL from the second PCR (Figure 1, step 5).

PCR Product Purification and Analysis

Amplification products from the third PCR were separated by gel electrophoresis on 1%-3% agarose gels, and individual products were gel-purified using QIAquick® Gel Extraction kit (Qiagen). When multiple bands were detected, all bands were isolated. The nucleotide sequence of each amplification product was determined using the transgene-specific primer 3 as a sequencing primer (Figure 1, step 6). Nucleotide sequencing was performed either at our DNA Core (University of Missouri-Columbia) or by SeqWright (Houston, TX, USA). The nucleotide sequences were aligned and examined to confirm the presence of the expected known transgene sequence and determine the flanking sequence representing the insertion site. The insertion site sequence was analyzed using Basic Local Alignment Search Tool (BLAST) (15) to determine the chromosomal location of the transgene.

RESULTS AND DISCUSSION

Lentivirus was used as a delivery system to create Lewis rat lines carrying an EGFP transgene (2). A total of nine transgene positive founder (F0) animals were recovered. While all nine founders were positive for the presence of the EGFP transgene, eight founders expressed EGFP based on epifluorescent microscopy of tail biopsies, while one founder (456) had no detectable fluorescence (Table 1). Our method for determining chromosomal integration sites was used to identify the chromosomal location(s) of the transgene in each of the nine founders. PCR products ranged in size from 100–1000 bp. Longer PCR fragments were generally necessary for determining the insertion site when the region contained repetitive elements. Several sites were successfully identified with recovered products as small as 110 bp, which included as little as 20 bp genomic sequence. For seven of the founders, we identified one to four integration sites depending on the founder. We failed to identify the integration site in two founders using PstI and HhaI digestion, and we did not pursue these further. It is possible that by using other restriction enzymes with 4–6 bp recognition sites or increasing PCR extension times to generate larger products, we would have successfully identified the integration sites in these founder lines. For seven lines, site-specific genotyping assays were developed for every integration site identified in the founder. In one case (463), we recovered genomic sequence that matched a sequence found on many chromosomes, so we could not assign a chromosomal location to this integration site. Nonetheless, the genotyping assay based on this sequence allowed the transgene integration site to be followed not only in the founder but in his offspring.

To follow segregation of the integration sites, the offspring from matings between each of the seven founders and wild-type Lewis rats were monitored for presence and expression of the transgene at each integration site using the site-specific genotyping assays and microscopic examination for GFP fluorescence. One founder (452) was infertile. For the remaining six founder lines, offspring were obtained, and both the insertion sites and the GFP expression were determined.

Founder 455 had two independent transgene insertion sites: one on chromosome 1, and a second on chromosome 5. When this founder was mated to a wild-type Lewis animal, two N1 offspring were recovered. One N1 carried the chromosome 1 integration site, but did not have detectable GFP expression. Lack of expression may have been due to positional effects. The other N1 carried the chromosome 5 integration site and did have GFP expression. The chromosome 5 transgene positive N2 offspring (n = 5) continued to have high fluorescence, demonstrating that GFP expression in this line was associated with the chromosome 5 transgene, and could be stably transmitted from generation to generation. This illustrates the importance of determining which transgene insertion site was correlated with GFP expression in order to successfully maintain a GFP-expressing line.

Line 456, which carried two transgene insertion sites, was unusual in that the founder did not display GFP fluorescence. Of the offspring recovered from this founder, three carried the chromosome 17 transgene insertion only and one carried the chromosome 9 transgene insertion only. While animals carrying the chromosome 17 insertion did not have detectable fluorescence, robust GFP fluorescence was seen in the animal that inherited the chromosome 9 transgene insertion. The chromosome 9 transgene positive N2 animals continued to have high fluorescence, demonstrating that GFP expression in this line was associated with the chromosome 9 transgene. We speculate that GFP expression was suppressed at the chromosome 17 insertion due to a position effect and that the founder was mosaic for the chromosome 9 insertion, resulting in undetectable GFP expression. In subsequent generations, inheritance of the chromosome 9 insertion was germline, and expression occurred in all cells leading to detectable fluorescence.

For line 458, three insertion sites were detected. By correlating GFP expression with inheritance of the various transgene insertion sites, it was possible to show that the chromosome 7 integration gave good GFP expression. This was confirmed in N2 animals (n = 10) carrying only the chromosome 7 integration. It should be noted that while none of the N1 animals carried the chromosome 10 integration site, this site was confirmed in the original founder by integration site-specific genotyping. It is possible that the founder was mosaic for the chromosome 10 integration site.

For line 459, which had a single detected transgene insertion on chromosome 2, two offspring were recovered that inherited the chromosome 2 transgene insertion site. However, neither of these animals exhibited GFP fluorescence. This could be due to a positional effect associated with the particular insertion site on chromosome 2 coupled with inheritance through the male lineage, or alternatively, we may have missed an insertion site in founder 459 that was associated with the fluorescence seen in the founder, which was not inherited by these two offspring.

Founder 463 had the greatest number of insertion sites; by continuing to correlate GFP expression with inheritance of specific transgene insertion sites, it was possible by the N2 generation to identify animals with single transgene insertion sites that maintained high GFP expression.

In addition to the lentivirus experiment described above, we have successfully used this technique to determine the lentivirus integration sites and to generate rat lines with single transgene integration sites for a Sprague-Dawley GFP transgenic (RRRC: 0065), derived from the line created by Lois et al. (2), and a rat model containing the human presenilin-1 gene (RRRC: 0061). To test whether our method for identifying insertion sites had broader application beyond just determining chromosomal locations of lentivirus transgene integration, we attempted to identify the transgene integration site of three additional rodent strains: (i) a mouse transgenic strain (MMRRC: 000366-MU/H) carrying the EGFP gene on the FVB inbred genetic background (16); (ii) a knockout mouse line (MMRRC: 000352-MU/H) involving the acetylcoenzyme A dehydrogenase long chain gene (Acadl), which carries a duplication of exons 3 and 4 with insertion of the neocassette into the approximately 11 kb intron 4 (17); and (iii) a transgenic rat line (RRRC: 0043), which contains a mutated version of the human HLA-B2705 gene on a Lewis genetic background (18). In the case of the FVB-EGFP strain and the Acadl knockout strain, we were able to determine precisely the insertion site on chromosome 3 and within the large Acadl intron 4, respectively. Our method failed for the HLA-B2705 transgenic rat line, which has a high transgene copy number (24 copies) in homozygous animals (18). The insertions are integrated at a single locus (18), and the multiple copies have probably integrated as concatamers.

A major advantage of determining the precise chromosomal integration site in animals created via pronuclear injection is that genotyping assays can be developed that allow animals heterozygous for a transgene to be easily distinguished from homozygous animals. Without this type of information, PCR-based genotyping assays can distinguish only whether animals carry the transgene. While it is possible to use Southern blot analysis and densitometry to measure transgene copy number as an alternative method to distinguish homozygotes from heterozygotes, it is laborious, timeconsuming, and not practical for many labs.

In summary, we have described a quick and straightforward method for determining the location of multiple chromosomal integration sites in animals created by lentivirus transgenesis. Our studies underline the need to carefully correlate a particular integration site with gene expression over multiple generations in order to create stable models. We have also found that this method has applicability for detecting transgene integration sites in other cases of random integration, but is probably limited to situations where very few tandem copies of the transgene have been integrated at the insertion site.

ACKNOWLEDGMENTS

E.C.B. and M.P contributed equally to this work. This work was supported in part by grants from the National Institutes of Health (NIH; U42 RR014821 and P40 RR16939). We thank James Sparks for technical assistance and Howard Wilson for assistance with graphics.

COMPETING INTERESTS STATEMENT

The authors declare no competing interests.

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Received 6 July 2006; accepted 22 August 2006.

Address correspondence to Beth A. Bauer or Yuksel Agca, University of Missouri-Columbia, Department of Veterinary Pathobiology, 1600 E. Rollins St., Columbia, MO 65211, USA. e-mail: bauerbe@missouri.edu; e-mail: agcay@missouri.edu.

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