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29 year old woman | Lancaster, TX, USA | Seeking men 25-35 within 50 miles
Loving Spirit, kind heart, great personality, strong morals, values and integrity. Great sense of humor. Just looking for somewhat the same
I am a beautiful intelligent woman seeking a wonderful individual. I am looking for someone with dreams and goals. Someone that even though they may not have yet accomplished their goals they are working towards them. I am a woman who wants things out of life. I am funny, laid back, spontaneous, outgoing, and lovable woman, I will try anything once that isn't too crazy. I don't have any kids but hope to have a family one day, I'm looking for someone to grow with. Someone who puts God first.
Coffee and conversation , Business networking , Cooking , Dining out , Fishing/Hunting , Movies/Videos , Museums and art , Music and concerts , Exploring new areas , Performing arts , Playing cards , Playing sports , Religion/Spiritual , Shopping/Antiques , Travel/Sightseeing , Volunteering , Watching sports , Wine tasting
Aerobics, Basketball, Billiards / Pool, Bowling, Cycling, Dancing, Football, Inline skating, Martial arts, Running, Skiing, Swimming, VolleyBall, Walking / Hiking, Yoga
I exercise 1-2 times per week.
I like Dogs, Horses
Some of my favorite local hot spots are a the dance theatre, museum, nice restaurant/lounge, to see a play, anything fun.
I love anything that can be done outdoors, I love being at the lake, watching a good classic movie, learning how to cook different types of food, listening to a good band, a variety of music, drama, mystery and romantic movies, traveling, Scandal.
Outdoor activities, Anything that has to do with water, Playing pool, Movies, Honestly I love doing everything, Anything fun, I also love trying new things, Cooking, Watching TCM movies, Going to listen to live bands,
A Boy name Dibs a book for my Psychology class.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'5" (165cms)
|5'5" (165cms) to 6'11" (210cms)
|Body type:
|Athletic and toned
|Slender, About average, Athletic and toned, Stocky
|Black, Blue, Brown, Grey, Green, Hazel
|Light brown
|Auburn / Red, Black, Light brown, Dark brown, Blonde, Dark blonde, Bald
|No Way
|No Way, Occasionally, Yes, but trying to quit
|Social Drinker
|Never, Social Drinker
|I'll tell you later
|No preference
|I'll tell you later
|No preference
|No preference
|Have kids:
|No preference
|Wants kids:
|Definitely, Someday
|Black / African descent
|Asian, Black / African descent, East Indian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian
|Christian / Protestant
|Christian / Catholic, Christian / Protestant, Christian / Other
Believe wholeheartedly in God and that he died on the cross for my sins. I fall short everyday but he still loves me and I will always return hone to my father and king.
|No preference
|Some college
|No preference
Business Administration


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Hospedaje El Virrey
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TripAdvisor Reviews Hospedaje El Virrey Pucallpa
Travel Blogs from Pucallpa
... and went to the market to do my food shopping for the week. Not much and nothing too exciting, few potatoes, rice, vegetables and 4-5 fruits, some tea bags and that's it. That would do my 2 daily meals. Here we go on a pirogue for a 45 minutes trip to reach Santa Clara, small village near Pucallpa where my maestro lives with his family. The people living there are Shipibos, name of the tribe.
We do the presentation, everyone seems nice and all the family live in ...
The Zoo with the Kids
... developed as the one here in Pucallpa. Still, I told myself that as long as I saw a leopard in the zoo that I would be happy.
I have never realized how much work it is being a teacher in charge of a group of kids. I was in charge of looking out for eight students in my group and they were all clamor and energy. First there was ...
Ucayali and the Bee
... humorous.
The spelling bee was initially christened with the marquee-catching name the "Ucayali Adventist School Orthographic Contest," but it was promptly changed to the "Ucayali Adventist School Spelling Bee" after I explained to Carmen that "Spelling Bee" is what such a contest is called in America. Besides, spelling bee sounds more ...
Evangelism, Dentistry, and Other Thoughts
... is tarnished. What I am getting at is that service that is interspersed with too much, say, evangelism, can have a negative effect if not executed properly and almost imperceptibly. But enough of that topic for now.
This week life around Km has been considerably quieter. This is due to the fact that six missionaries have been sent to Km 38 in country to work on the "ranch." Each week ...
The Storm and the Sludge
... their teeth with water that comes directly from the tank, but I do not trust the quality on account of the adverse effect that have already befallen me from drinking camu camu drink from a street vendor.
Naturally, due to the newfound extensive use that the filter is being placed under due to the addition of 17 souls at Km 8, it becomes necessary to clean it out frequently. I took it upon myself to clean the ...

subset: commoncrawl


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Data on 119 preschool children ages 3 to 6 revealed normal-weight and overweight or obese participants did not show significant differences in physical activity levels, suggesting that exercise rates may not be an important factor in childhood obesity. However, researchers found children with lower socioeconomic status had greater BMI and media consumption. The findings were published in PLoS One.
View Full Article in:FoodNavigator
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The Comedy In Fifty Shades of Grey
By Amy Nicholson
Wild Tales Lays Bare Everyone's Awfulness
By Stephanie Zacharek
Approaching the Elephant Is a Bracing Education Doc
By Calum Marsh
By Stephanie Zacharek
By Inkoo Kang
The Results of Our 2014 Film Critics' Poll
By Voice Film Critics
By Alan Scherstuhl
The Blue Room (La Chambre bleue)
By Alan Scherstuhl
Though it offers an invitation that cannot be fulfilled, the great Dietrich shot is, if anything, friendlier and more forgiving than that of Zhang's nightclub escort Bai Ling, who aims her scornful stare at anyone with the gall to ogle or proposition her. Taken out of context, Bai's defiance, its power enhanced by the low angle, could be invoked as a contemptuous post-feminist dismissal of the "controlling male gaze" identified by Laura Mulvey 30 years ago, though the movie itself reveals that Bai unconsciously desires to be trapped by her sadomasochistic longings for embittered playboy Chow Mo Wan (Tony Leung).
There she stands then, in a spangled black cheongsam, a noirish totem of sexual aloofness, in her room, 2046, at Hong Kong's Oriental Hotel. Her upper lip is cast in shadow as it separates provocatively from its neighbor. Her neatly coiffed head is cocked slightly to her left at an angle that would seem quizzical if it didn't seem she knows all the damn answers (in fact, she has none). She has, meanwhile, arrayed herself in insolent contrapposto: Her right hand is spread on her right hip in such a way that it crooks the arm at a 90-degree angle at the elbow; her left hand caresses her abdomen with the scarlet-tipped fingers at 10 o'clock (much too early for bed in mid-'60s Hong Kong). This accentuates not the curve of her back, as the New York Times review headline euphemistically put it, but the prominence of her bust, which must be pressing painfully against her too tight sheatha clear mark of masochism. The pose echoes Dietrich's akimbo stances in The Blue Angel and especially Sternberg's 1932 Blonde Venus. It's an advertisement, a challenge, and a taunt.
Like the Dietrich photo, the Zhang still is not a film frame but a production shot. Taken by Wing Shya, the celebrated Hong Kong photographer and graphic artist who works on most of Wong's movies, it was published in the Voice, the Daily News (a decision I was involved in as an editor there), and the Times, where it took up a whopping 115 inches of prime real estate on the first page of the August 5 Weekend Arts. It also graced the cover of the JulyAugust issue of Film Comment and is one of 13 images collaged on the U.S. film poster, where it was reproduced in a panoramic version that shows more of room 2046 than that version offered to the press on the Sony Classics website. On the poster, it sits above another image of Bai looking out at the viewer, but this time lying naked in bed and looking suitably vulnerable after her seduction by Chow: The latter image delivers a sadistic lash to the Zhang fancier, but her haughty expression and fetishizing clothes in the top still draw the eye more than her glowing skin in the lower.
Bai's shrouded eyes, announced by scimitar brows, stare imperiously down at . . . who or what? Chow, who in the movie peeps at her through a hole in the thin wall that separates their rooms? Wong? Wing? Us? All of the above, but most urgently herself. During the mesmerizing 27-second, eight-shot slo-mo sequence in the film (at the start of which Wing probably took the photo), Bai flits around her room getting ready to go out and earn her living. She adjusts the constricting collar of her dress, sips from a glass, puts on earrings, and assesses herself before mirrors. Her rapt narcissism, born of insecurity, and her na doom her to fall unrequitedly in love with Chow. An ominous counterpoint to her proud self-appraisal of her allure is Connie Francis's aching 1960 rendition of the rumba "Siboney" that ushers in the sequence: Written in 1929 by the Cuban composer Ernesto Lecuona and also heard in Fellini's Amarcord, the song is a lament for a departed lover "If you do not come back I will die of love"that augurs Bai's journey into romantic torment.
Take a bullet train to the year 2046, or even to the next five minutes, to recover the movie memories you've mislaid, and you may find yourself hurtling into a morass of self-deception. In cinema, as in life, the velvet light trap of the mind imperfectly recalls the images that flickered by at 24 frames per second so that we re member nothing we saw on-screen the same way twice, any more than we exactly remember experiences. As Mary Ann Doane wrote, interpreting Freud, in Femme Fatales (1991), "Memory is a palimpsestthe sum total of its various rewritings through time. The 'event' which is remembered is never really accessible as such." We are our own Rashomons, our own Wong Kar-wais.
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Oceanfront 4BR/2BA. 2 Oceanfront decks. Large kitchen with Ocean view. Deck off master bedroom. Frieze carpet, 16' tile, Energy Effeciency Award plus spray foam insulation. Anderson windows, hot tub, tankless water system.Security system, Trane Variable Speed Air Handlers, bamboo floor in dining room, brick paver driveway and covered parking attached to house. Must see to appreciate all the 'extras'. Price just reduced $100,000 to $650,000. July 27, 2012.
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Speaking at Chautauqua
FRIDAY: Leslie Berlin, historian with the Stanford University Silicon Valley Archives, will discuss the contemporary American West and the Silicon Valley technology boom. 10:45 a.m.
MONDAY: A theme week on the rise of Brazil as a world superpower begins with a lecture by award-winning National Geographic photojournalist Tyrone Turner. 10:45 a.m.
WEDNESDAY: Brian Winter, Thomson Reuters Brazil correspondent and author of "Why Soccer Matters," will speak about the importance of sports in Brazil. 10:45 a.m.


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10 killer photography tips the pros won't tell you
Photography Tips From The Pros: Focus stacking
Focus stacking is a neat trick that allows you to create images with very wide depth of field so the whole image is sharp, and it's especially useful when shooting macro subjects.
The subject needs to be motionless and the camera mounted on a tripod because the first step in the technique is to take a sequence of shots with the focus at different distances.
Take the first shot with the nearest part of the scene in focus and then, without moving the camera, refocus just a little further in and take the second shot.
Repeat this process of refocusing further into the scene before each shot until the furthest part of the subject has been photographed in focus.
The next stage is to combine all the images. While this could be done manually using any image editing software that supports layers it would be very time consuming.
Fortunately, Photoshop's Photo Merge function is capable of doing the work for you, or you can download Combine ZP for free.
Famous Photographers: 100 things we wish we knew starting out
How to see photos like famous photographers… every time you shoot
Best camera focus techniques: 10 surefire ways to get sharp photos
Getting sharp images: every photo technique you need to know starting out


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Lamar and the Taylor farm experiment
In the early 1880s, L.Q.C. Lamar was thinking about retirement from public life. He started purchasing land in the small town of Taylor, just south on Oxford. By 1882, he had acquired 550 acres along the Mississippi Central Railroad. On a hill overlooking the farm, he had a small wooden frame home built in the New England farmhouse style. There were also barns and shelters for the stock, and a dairy barn.
He wrote to his farm manager, William Knight, "that place is to be my future home at the end of my services here, I shall go to that place and spend the remainder of my life. You see, therefore, that you are not merely in charge of a nice farm and fine stock, but that you will contribute much to my future comfort." (October 22, 2010, Page 2B)
Solitude: Lamar and agriculture
L.Q.C. Lamar is known nationally as a congressman, senator, Secretary of the Interior and associate justice of the United States Supreme Court, but he is not known for his interest in, and ownership of, farming operations. In 1854, Lamar was living in his home state of Georgia. He had established a law firm in Macon but he did not have any use for his extensive slave holdings. (October 15, 2010, Page 3B)
'Oxford in the Civil War' – A new book from History Press
"Oxford in the Civil War: Battle for a Vanquished Land" by Stephen Enzweiler is the new book just published by The History Press of Charleston, S.C. The author is a journalist and senior editor for "Y'all" magazine published here in Oxford and he writes extensively about Mississippi and the South.
I really didn't find out anything that I didn't already know, but the way the author has presented the data makes for pleasurable reading. I have read these stories over the years in various different places, but Enzweiler presents them in manner that follows Oxford from its earliest day through the war years. (October 8, 2010, Page 2B)
Vaught, Bryant meet for the first time as head coaches in 1947
The Homecoming game on Sept. 20, 1947, would be the first time Ole Miss football coach John H. Vaught and Kentucky coach Paul "Bear" Bryant would meet as head coaches. Over the next 25 seasons, they would meet again and again as head coaches in the SEC. Oxford and Ole Miss historian Jack Lamar Mayfield takes a closer look at that 1947 game. (October 1, 2010, Page 2B)
Superhuman faith and tenacity in the face of inhuman adversity
This week, local historian and columnist Jack Lamar Mayfield writes about a young man from Alabama who made Ole Miss his home and forever became an honored member of the Ole Miss family — Roy Lee "Chucky" Mullins.
It was just a short 21 years ago that Chucky was a vibrant, 20-year-old member of the Ole Miss football squad. He had worked his way onto the Ole Miss team with exceptional ability that Coach Billy Brewer had seen when he was first introduced to Chucky. (September 24, 2010, Page 2B)
The Commodores and the Rebels: More than 11 decades of history
Local historian Jack Lamar Mayfield takes a look back at the Ole Miss-Vanderbilt football series, writing about the first few meetings between the two schools and then taking a look at a notable 1947 meeting when John Vaught's Rebels overcame an early-season loss to the Commodores to win the Southeastern Conference championship. (September 17, 2010, Page 4B)
30,000 Yankees encamped at College Hill
Stories of courage and honor surround the Yankee's occupation in the College Hill area after Gen. Ulysses S. Grant crossed the Tallahatchie River near Abbeville and went on to Oxford.
Grant's second in command was Gen. William T. Sherman. He had crossed the Tallahatchie at Wyatt's Crossing, just to the west of Abbeville, and had moved his 30,000 troops into the area around College Hill. (September 10, 2010, Page 3B)
The origin of Ole Miss football
Saturday, Nov. 11, 1893, was the first time an organized football-game was played by students of the University of Mississippi. Oxford EAGLE columnist Jack Lamar Mayfield takes readers back to the scene of that first game coached by Dr. A.L. Bondurant. (September 3, 2010, Page 5B)
The story of North Mississippi College
This week Oxford EAGLE columnist Jack Lamar Mayfield gives readers another lesson Lafayette County Education. Last weeks column he wrote about the formation of a new state university in which the Mississippi Legislature voted for Oxford to be the home site. This week, is about another another institution of higher learning that preceded the University of Mississippi in Lafayette County. (August 27, 2010, Page 2B)
James Alexander Ventress: The Father of Ole Miss
Mississippi House of Representatives member James Alexander Ventress, in early February of 1840, introduced a bill "to provide for the location of the State University." He was chairman of the house committee on the seminary fund. The House passed the bill on Feb. 10 and then sent it to the state Senate. The Senate quickly passed the bill and sent it on to the Gov. Alexander G. McNutt, for him to sign into law. He signed the bill on Feb. 20, 1840. (August 20, 2010, Page 3B)


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- Home
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Wings of Faith provides on-going support and donations to missionaries mainly serving Native American populations in the United States and is committed to helping the poor, abused, hungry and forgotten people in these communities. Our organization utilizes a team of volunteers to pilot light aircraft and drive ground vehicles to spread the gospel of Christ in tangible ways by transporting Bibles, food, clothing, medical supplies and other necessary aid to those who need it most.
For over 35 years, Wings of Faith has provided support and charitable donations to missionaries serving Native American populations in Mexico, Central America, and the western United States. We use light aircraft and ground vehicles to spread the gospel of Christ in tangible ways … getting Bibles, food, clothing, medical supplies, and other necessary aid to those who need it most.
Just released article from the Orange County Register. Read more about it here
One of the first Indian reservations that I served on was in Holbrook, Ariz. That was 40 years ago, and Wings of Faith has been ministering to the... Read More
The hangar distribution center we're building in Holbrook, AZ will help us serve our missionaries and thousands of indige-nous people... Read More
Thanks to the Lord's help we have been ex-panding and growing this project for several years now. In 2014 we delivered 3500 back-packs with... Read More
Rebar installation... Read More
More pictures coming in, materials for... Read More
Chris DeJong, a lovely woman and artist, is the master craftsman behind the Gleanings' murals, which are displayed in the Dining... Read More
More pictures coming in, foundation... Read More
Please follow this link to read the entire story from the Rapid City... Read More
During these tough economic times, American Indian families across the country are having trouble saving extra money for school supplies for... Read More
Donate to the W.O.F. Hangar Project Fund Thank you to everyone who is making the Wings of Faith hangar at Holbrook, Arizona possible. This has... Read More
America's forgotten... Read More
4/8/2015 This little girl and her grandmother live in this one room, plywood house along with her parents and siblings, no water, no... Read More
Want to help by displaying this flyer at your church or office? Download PDF here Wings of Faith Ministries has served the Native... Read More
Dear friends and family, As many of you are already aware, Jodie and I have been accepted as missionaries with Mission Aviation Fellowship,... Read More
Dave Snelling 1947-2015 Loving husband and devoted father. We are honored to keep the memory of Dave alive... Read More
We were further blessed with over 8,000 lbs. of soup and trail mix from the Youth with a Mission folks at Gleanings for the Hungry near Fresno, CA.... Read More
Blessings abound again at the Wings of Faith storage areas. We received 8 full pallets of coffee, tea and Lipton soup mix last week from Feed the... Read More
Tom Leedom, President Joe Terry, Vice President Carol Leenert, Secretary Jim Yost,... Read More
Wings of Faith Ministries Inc. Take Off Points Monthly Breakfast Meetings Resume in January WOF monthly breakfast schedule takes a brief... Read More
With the holidays and cold weather upon us, Stussy clothing made a great donation on the 18th of Dec. of some wonderful clothing! Included were cold... Read More
What about Christmas in September? You may have heard about our annual Christmas Airlift, when we deliver Christmas gifts to poor children... Read More
These pictures were taken at Tustin Acura. Girls are SADD volunteers who have collected app 200 toys to Wings of Faith to be donated to NAI Christmas... Read More
On Monday, Nov. 24 we were able to deliver 6 pallets of Christmas goodies and clothes to Yuma for Pastor Alex Montoya and Mike Claussen to use in... Read More
"I was naked and you clothed me." Those haunting words of Jesus refer to our works of mercy toward... Read More
From Stone Equipment in Corona, we now have some help at the airport, a small but fairly agile forklift! Stone equipment had already put on new... Read More
Christmas Airlift Deliveries Begin December 1st. We seek your prayers, support and encouragement as this year is a really big year for the Airlift... Read More
God bless you brothers and sisters at Wings of Faith. This is Jose just want to thank you guys for for all the stuff you guys bring us . Brother dale... Read More
Dale will speak about his retirment, plan for the future and vision for Wings of Faith Also, we will discuss plans for distribution for the... Read More
Thank you Partners for are making it possible for us to continue to provide food for people that are starving, help clothe children that are in... Read More
The event at chino airport it will take the place of our sept breakfast and we will have a display of the cessna 206 and a booth in the organization... Read More
School Supplies in the Hands of Young Students/Christmas Airlift Campaign Kickoff Original Web Version Read... Read More
Distributing backpacks and school supplies to less fortunate families and... Read More
Wings of Faith is busily preparing for the 2014 Christmas Airlift. This is one of the biggest outreaches each year for WOF and we would like to ask... Read More
Bringing hope love and toys during this Christmas season to less fortunate families and... Read More
Outreaching to the people of Fresno California with much needed... Read More
Download the newsletter here... Read More
On Saturday, June 7, Wings of Faith held its first open house in several years, this one centered around the airport operations area and the aircraft... Read More
Dear Dale Whinery & Staff: c/o Wings of Faith Headquarters Hope this letter finds all you California folks in good health... Read More
We would appreciate the opportunity to share with you our ministry and what God has challenged and equipped us to do. Please join us for a day of fun... Read More
Feed the Children (see feedthechildren.org, donated two pallets of breakfast cereal to be used by the folks on the Native American Reservations this... Read More
On Feb 11, Jason Ramirez from Wings of Faith was able to pick up 13,000 lbs of food for our missions at Gleanings(gleanings.org) headquarters in... Read More
The Christmas Airlift 2013 is being helped along in a big way by Walgreen's Pharmacies in Orange County, CA. The area manager has opened four... Read More
The container was loaded yesterday for the Phillipines, thanks to many of you who contributed with goods or dollars! Wings of Faith was able to fill... Read More
All Tribes Sonlight Church Needs a New Roof UPDATE: Special video from All Tribes Sonlight Church from Wings of Faith on Vimeo. A/C... Read More
This was an event put together last year or so in Nogales Mexico. They went into the poorest areas and recruited kids and thier families to... Read More
Do strange events in recent headlines mean something is up spiritually, or simply the sign of the times or mere coincidence? We had a close encounter... Read More
Once again Costco has blessed Wings of Faith with a wonderful donation. It is at this time of year that Wings of Faith starts to prepare for the... Read More
Yesterday, Sept. 11, we received over 3,000 backpacks from Obey Clothing, see ww.obeyclothing.com. This will complete the school term for fall of... Read More
Sometimes we feel a little overwhelmed by what God is doing. It seems the Lord is blessing us to be a blessing by supplying us with unique and... Read More
We are nearing the completion of the backpack and school supply project for this year! God was very gracious to us and supplied funds to purchase and... Read More
A large contingent of missionary aviators from around the globe attended EAA Airventure in Oshkosh, WI. The Airventure runs for seven days, usually... Read More
To be a blessing. Through our partners at Serve-One, we can bless others with healthy and nutritious food. Seniors with digestive problems are... Read More
School Supply Project in Full Wing and Wheel WOF started delivery of over 1,000 backpacks, binders, notebooks, pencils, etc with a... Read More
Pilotsafety.org invited Wings of Faith to have an information table at an all-day aviation safety seminar on Saturday in Long Beach. We were able to... Read More
This gallery shows the Christmas... Read More
Click pictures for... Read More
Wings of Faith representatives and members of the Christian Family Center in Corona, CA, (led by Pastor Julio Romano) converged on the monthly... Read More
Thank you all so much for your help in caring for our tribes. God bless you! Susan... Read More
Yes, our Lord knows needs and how to fill them! Serve-One in conjunction with Operations 29:11 and Wings of Faith were able to supply 5,000 lbs.... Read More
Compliments of Serve-One and Operations 29:11 , the jointly operated warehouse in Holbrook now has a way to store fresh food until... Read More
During Holy Week, Serve-One(see serve-one.org) contacted Wings of Faith with the opportunity to provide hams, yams and butter to those in need.... Read More
Starting tomorrow, March 26, Wings of Faith will begin distribution of some school supplies provided by Feed the Children(see... Read More
The school that Thoreau Navajo Outreach worked with this year was a rural mountain boarding school called "Dibe Yazhi... Read More
On January 21/22, 2013, we witnessed an unprecedented meeting of over 60 representatives of various churches, non profits and ministries serving... Read More
Last week Wings of Faith, in cooperation with Operation 29:11(operations2911.org) and Gleanings(gleanings.org), was able to distribute 20 barrels... Read More
First week in January, Stussy clothing came through to the disadvantaged living in the colder parts of the southwest United States on the Native... Read More
The Wings of Faith Ministries Christmas tradition was entirely and completely a success in 2012, with over 8,000 gifts, games, blankets and winter... Read More
Wings of Faith Ministries Inc. received official notice this week that we now linked with Feed the Children... Read More
Posted on 08-22-12: You just returned from attending the EAA 2012 AirVenture at Oshkosh the last week of July, where you set up a booth in the... Read More
Tribes4Christ sent this ecard to Wings of Faith. If you have partnered with us in any way, whether in prayer, financial support or sweat equity, this... Read More
Posted by: Tom Leedom 8-9-2012 For those that may not be familiar with Gleanings for the Hungry: Gleanings is a YWAM organization... Read More
Posted by Bruce Nelson 07-26-2012 I received a phone call from Wings of Faith board member Jim Yost on June 23rd asking if I could help out a... Read More
Posted by Joy Lais 07-21-2012 We're on our way, flying off into severe clear skies for our annual trek to Oshkosh, WI for the EAA AirVenture... Read More
Posted by Dave Cheney 7-5-2012 On June 7, 2012, my wife Debbie and I made a WOF missionary flight from Southern California to Window Rock Arizona;... Read More
Effective partnerships are essential to the WOF ministry. An example of great cooperation and teamwork is the partnership we have with Gleanings.... Read More
Posted on: June 14th, 2012 By: Bruce Nelson I awoke early Friday, June 8th, in anticipation of a flight to Polacca, Ariz. (75 miles northeast... Read More
Posted on: June 13th, 2012 War on Hunger seems to be a never ending war. Traveling the world as a missionary for more than 40 years, I have... Read More
We have just received a Cessna 152 to use in our Missionary Pilot Training Program! Thank you, Jesus! We are also looking for two Cessna 182... Read More
To read our latest newsletter online, click here. If you'd like to receive our newsletter via U.S. mail, send us a note... Read More
What can a single volunteer do? More than one can imagine! Our volunteer pilots, drivers and warehouse workers rise to the challenge and deliver.... Read More
Meeting Place for Fellowship Breakfast
Thank You
Wings of Faith Ministries
Donate to Dave's Memorial Fund
We are $53206 of the way to our goal of $100000
Learn more on how to help the hangar project


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Welcome to the Zimmerman Research Group webpage. Our group specializes in the development and application of first principles methods to solar and catalysis challenges. Please see the above links for specific descriptions of our ongoing work. Also feel free to contact Dr. Zimmerman at paulzim.at.umich.edu with any questions.
Recent Publications
1. P. M. Zimmerman, "Single-Ended Transition State Finding with the Growing String Method," Journal of Computational Chemistry, published online: Link.
2. Z. Sun, G. A. Winschel, P. M. Zimmerman, P. Nagorny, "Enantioselective Synthesis of Piperidines through the Formation of Chiral Mixed Phosphoric Acid Acetals: Experimental and Theoretical Studies," Angewandte Chemie International Edition, 53, 11194-11198 (2014). Link. Highlighted in SYNFACTS October 2014: Link.
3. P. M. Zimmerman, "Reliable Transition State Searches Integrated with the Growing String Method," Journal of Chemical Theory and Computation, 9(7), 3043-3050 (2013). Link.
4. P. M. Zimmerman, "Navigating Molecular Space for Reaction Mechanisms: An Efficient, Automated Procedure," Molecular Simulation, Special Issue: "Recent Advances in the Molecular Simulation of Chemical Reactions" DOI: 10.1080/08927022.2014.894999 (2014).
5. P. M. Zimmerman, "Growing string method with interpolation and optimization in internal coordinates: Method and examples," Journal of Chemical Physics, 138, 184102 (2013). Link.
6. P. M. Zimmerman, "Automated Discovery of Chemically Reasonable Elementary Reaction Steps," Journal of Computational Chemistry, 34(16), 1385 (2013).
7. P. M. Zimmerman, C. B. Musgrave, M. Head-Gordon, "A Correlated Electron View of Singlet Fission," Accounts of Chemical Research, 46(6), 1339-1347 (2013).
8. P. M. Zimmerman, D. Tranca, J. Gomes, D. Lambrecht, A. T. Bell, M. Head-Gordon, "Ab Initio Simulations Reveal that Reaction Dynamics Strongly Affect Product Selectivity for the Cracking of Alkanes over H-MFI," Journal of the American Chemical Society, 134, 19468-19476 (2012).


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Duchenne Muscular Dystrophy (DMD) is an X-linked genetic disorder that results in the production of a dysfunctional form of the protein, dystrophin. The mdx5cv mouse is a model of DMD in which a point mutation in exon 10 of the dystrophin gene creates an artificial splice site. As a result, a 53 base pair deletion of exon 10 occurs with a coincident creation of a frameshift and a premature stop codon. Using primary myoblasts from mdx5cv mice, single-stranded DNA oligonucleotides were designed to correct this DNA mutation.
Single-stranded DNA oligonucleotides that were designed to repair this splice site mutation corrected the mutation in the gene and restored expression of wild-type dystrophin. This repair was validated at the DNA, RNA and protein level. We also report that the frequency of genetic repair of the mdx mutation can be enhanced if RNAi is used to suppress expression of the recombinase inhibitor protein Msh2 in cultures containing myoblasts but not in those heavily enriched in myoblasts.
Exogenous manipulations, such as RNAi, are certainly feasible and possibly required to increase the successful application of gene repair in some primary or progenitor muscle cells.
Duchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder with an incidence of 1 in 3,500 live born males. It is caused by the dysfunction of dystrophin (DMD), a large 427 kDa cytoplasmic protein that forms a link between actin filaments and the dystrophin-glycoprotein (DGC) complex [1]. The loss of dystrophin is thought to affect the integrity of the cell membrane leading to a disruption in cellular calcium homeostasis and eventually cell necrosis [1-3]. At the genetic level, one-third of boys afflicted with DMD have point mutations, insertions or deletions while the remaining two-thirds have gross deletions or rearrangements within the 2.5 Mb gene [4] [OMIM #310200 Muscular Dystrophy, Dystrophy Type, DMD] and all generally cause a translational frameshift.
Therapeutic strategies such as gene replacement have been used to restore protein expression by effectively re-engineering dystrophin cDNA. In addition, attempts have been made to introduce smaller functional homologs of DMD to cells in an attempt to reverse the muscle degradation process [5-8]. Vectors, which carry these constructs, however, often elicit an immune response [8,9]. An RNA/DNA chimeric oligonucleotide (chimera) has been used successfully to correct the splice site mutation to generate a genetic alteration in the dystrophin gene, in both ex vivo and in vivo studies in mouse and dog models of DMD [10-13]. In a pioneering study, Bartlett et al. [10] showed the correction of the canine dystrophin gene within skeletal muscle fibers after injecting the chimera into the cranial tibialis of Golden Retrievers. The production of wild-type dystrophin was restored and could be detected for up to 48 weeks at the site of the injection. Rando et al. [11] demonstrated chimera-directed repair of the dystrophin gene in the mouse extending the results from the dog model; once again the corrected cells were again localized to the injection site.
Antisense oligonucleotides also have been used to alter the splicing of the dystrophin message in a process generally termed, exon skipping [14-16]. A large number of exciting reports have demonstrated the feasibility of altering the splicing patterns of the mutant DMD transcript [15,17-21]. While this approach is likely to have significant therapeutic benefit, the original DMD mutation(s) remains and thus a continuum of antisense ODN delivery will be required [22].
When muscle fibers are damaged within the body, a population of quiescent precursor muscle cells, or satellite cells, become activated myoblasts [23,24], rapidly divide and initiate the regeneration of the injured muscle [24]. In the case of DMD, the muscle is thought to be constantly going through cycles of degeneration and regeneration due to the instability of the musculature and, therefore, abundantly populated with myogenic precursor cells. Thus, an important long term therapeutic approach then would be to isolate and genetically correct the myoblasts at the DNA level with subsequent transplantation to the animal model in order to restore functional dystrophin to the muscle fibers.
We have begun to develop a more defined ex vivo strategy in which modified single-stranded oligonucleotides are designed to repair the mutant dystrophin (ex vivo) in cell cultures enriched in myoblasts. A current challenge is to establish a frequency of repair that leads to a level of dystrophin expression that can be detected in targeted myoblasts. Previously, Bertoni et al. [25] have used fluorescently labeled oligonucleotides to correct a point mutation in cells derived from the dystrophic mdx5cv mice. We now repeat the correction of Dmd point mutation using unlabeled modified single stranded oligonucleotides and establish novel quantitative assays to measure the dystrophin production in myoblasts. We also stimulate the correction reaction by using RNAi against Msh2, a known inhibitor of the gene repair reaction [26] and observe higher levels of gene repair.
Isolation of primary cultures from dystrophic mdx5cv mice containing myoblasts with determination of isolation efficiency
In the natural muscle environment, satellite cells rest in a quiescent state along the periphery of an intact muscle fiber. When muscle fibers are damaged, muscle precursor cells, referred to as myoblasts, divide and fuse with the damaged muscle fibers to repair them [see [27] for review]. In order to isolate the muscle precursor cells for gene repair experiments, muscle tissue was removed from the limbs of 1 to 3 days old mdx5cv mice, enzymatically digested and plated. A pre-plating technique was used to purify these cells from fibroblasts present in the explant [27,28]. It is known that the fibroblasts adhere to the plate rapidly, usually within 2 hours, and, therefore, the pre-plating technique is used them from the culture by passaging the supernatant at a 2 hour time point (PP1) and again at a 24 hour time point (PP2) [28]. Thus, the supernatant is passaged until PP4 at which time cells are harvested to determine the efficiency of myoblast isolation by incubation with an antibody against desmin (expressed in myoblasts). We estimate the efficiency of myoblast isolation in this experiment to be 53% as determined by counting the number of desmin positive cells and dividing this number by the number of total cells observed in multiple fields of view (data not shown). The remaining 47% are most likely fibroblasts. Thus, in the experiments outlined below, we utilize cultures that contain approximately 50% myoblasts and 50% fibroblasts. Primarily myoblasts are a target because only corrected myoblasts that fuse to form myotubes will be capable of expressing corrected dystrophin.
In order to determine the efficiency of gene repair of the chromosomal dystrophin gene in myoblasts, it was important to determine the level of transfection of oligonucleotides. Oligonucleotides in varying amounts were complexed with Fugene 6 to observe the levels of uptake within the myoblasts. Fugene mediated transfection only showed a 4–6% uptake of the FAM-labeled oligonucleotides (data not shown). Therefore, we chose to use jetPEI, which is formulated specifically for delivery of oligonucleotides to deliver the ODN into the primary cultures. Myoblast cultures were seeded at increasing densities and lipoplexes composed of 6 μL of jetPEI and 3 μg of ODN were added to cultures with readout of uptake by FACS analysis after 24 hours. There was no difference in the amount of oligonucleotide uptake at different cell numbers as shown in Figure 1 (average uptake, 36%). FACS was used to quantify uptake of FAM-labeled oligonucleotide as well as cell death by propidium iodide. Thus, a range of cell densities can be transfected using the jetPEI complex.
Figure 1. Optimization of ODN uptake in primary myoblast cultures from mdx5cv mice. The percent of FAM labeled ODN uptake complexed with the transfection reagent jetPEI (blue bars) as a function of the seeding density of the myoblasts is shown as well as the amount of background fluorescence when myoblasts are incubated with either the FAM oligo or jetPEI alone. The cell density is the 2.5 × 106 when the cells are treated with FAM or jetPEI alone. The amount of cell death at each cell density and control samples is also shown (red bars).
Gene repair of dystrophin directed by modified single-stranded oligonucleotides in purified myoblasts from mdx5cv mice
The mdx5cv mouse has a point mutation in exon 10, which creates a splice donor site that results in a 53 base deletion within the exon (see diagram in Figure 2). This deletion results in a reading frameshift wherein a premature stop codon is created 95 bases downstream of the mutation site. If the ODN directs correction of the base, then the correct splice pattern is recreated and the 53 deleted bases remain in the message; in effect, restoring the reading frame. To evaluate the restoration of the 53 base pair insertion, a reverse primer was designed to bind to the deleted portion of exon 10. Thus, amplification can only occur if the targeted splice site corrected and the full-length mature message is produced at detectable levels. To determine the level of expression of dystrophin mRNA where the deleted region had been restored, mRNA was reverse-transcribed using a primer specific for exon 12, which is present in all of the transcripts, corrected or not.
Figure 2. Mutation in dystrophin gene of mdx5cv mice and correcting ODNs. Mdx5cv mice have a single point mutation (A to T) in the dystrophin gene, which is both bolded and underlined in the gene sequence above. This single base mutation will create a donor splice site within exon 10 and cause a 53 base portion of this exon to be spliced out of the mature transcript as depicted by the dashed line in the diagram. A frameshift results in a stop codon in exon 11 (*) and will ultimately result in the production of a truncated dystrophin protein. Because the natural donor site is still present in the transcript there is a possibility that some full-length message, containing exon 10 is made and this is shown as the solid line. The ODNs, mdx47T and mdx47NT, shown above, were designed to correct the substitution mutation and the target base is underlined.
We outlined oligonucleotide-mediated gene repair reactions containing ODNs, mdx47NT or mdx47T, designed to correct the T substitution mutation within exon 10 of the dystrophin gene. These are depicted in Figure 2. For clarity, we provide the wild type and mutant DNA sequence, as well. The "T" indicates that the 47-mer is designed to target the transcribed strand while the "NT" refers to a molecule designed to target the non-transcribed strand.
The oligos were delivered to myoblasts by jetPEI and the cells were allowed to recover for 24, 48, 72 and 96 hours respectively at which point genomic DNA was extracted. Twenty-five nanograms of DNA were subjected to absolute quantification using the Taqman probe analysis. This assay measures the amount of wild-type or corrected dystrophin DNA within each sample. The probe is designed to recognize only dystrophin sequences that contain the changed base and, thus, no direct sequencing of the gene is necessary to confirm sequence alteration [29]. Figure 3A shows that wild-type dystrophin can be detected by 24 hours after transfection with either mdx47NT or mdx47T. The highest amount, 331 pg, a significant 15.72 fold increase (p < 0.001), is detected when cells are targeted with mdx47NT. mdx47T corrects less efficiently with an overall average of 59 pg of wild-type dystrophin detected from 24 to 96 hours, indicating that there is a strand bias toward the nontranscribed strand of the dystrophin gene (Figure 3). PCR products directly from the probe assay were sequenced to confirm the correction within genomic DNA and the sequencing results show that the corrected base (A:red peak) is the major product in the reaction with a very minor peak (T:green peak) being the uncorrected mutant base. This minor mutant peak correlates to the specificity of the probe reaction in that a low level of the mutant base is detected, but as shown in Figure 3A, this background is not statistically significant in the quantitation assay.
Figure 3. Quantitative measurement of dystrophin correction in primary cultures containing mdx5cv myoblasts at the DNA level and sequence analysis. A: Total genomic DNA was isolated from targeted myoblasts at various time points, 24 to 96 hours after introduction of ODNs, mdx47NT or mdx47T. Twenty-five nanograms of this DNA was subjected to absolute quantification by PCR analysis with a FAM labeled probe specific only for the wild-type dystrophin sequence. A standard curve had been previously generated using C2C12 (WT) genomic DNA and the amount of wild-type dystrophin has been extrapolated from this standard and is presented as nanograms of wild-type dystrophin. These data represent the average of three experiments that were analyzed in triplicate. Significance was measured by a Student's t-test and samples were considered significantly different with a p value of less than *0.05, **0.01, ***0.001. B: DNA sequence reaction of PCR products showing the chromatogram representing a mixed population at the targeted base (base 102) with adenine (green/corrected base) being the major peak and with thymine (red/mutant base) being a minor population within the reaction.
To evaluate gene repair at the RNA level, quantitative RT-PCR was carried out using a primer set specific for exon 9 and the deleted portion of exon 10. Table 1 illustrates the cycle number at which the restored portion of the exon initially appears. In these analyses, higher Ct values are equivalent to a lower amount of corrected message while a lower in Ct values signifies an increase in the presence of the deleted portion of exon 10. Table 1 shows that the deleted region is detected at 72 hours of targeting followed by 96 hours in DM. A statistically significant 6-fold increase in message is detected above nonspecific levels. Taken together, the data indicate that the 47NT is capable of correcting the dystrophin gene and restoring dystrophin expression to the targeted population. Interestingly, the fold increase in correction detected of the dystrophin gene detected with the Taqman probe (15.72×) (see Figure 3A) is not equivalent to the fold increase in expression (6 fold). A likely explanation for this observation indicates that some correction is occurring in fibroblasts, which do not express dystrophin, although such a comparison may not be possible.
Table 1. Quantitative measurement of dystrophin, GAPDH, and CKM.
The donor splice site in the DMD gene in mdx5cv mice causes a frameshift which results in a translation of truncated dystrophin protein (10 of the 79 exons). As shown above, correction of the mutation at the DNA level resulted in the restoration of intact dystrophin message. To determine whether the corrected mRNA is translated into normal dystrophin that contains exon 11, cells were seeded and once again transfected with mdx47NT or mdx47T under conditions that provided the highest level of gene correction. The entire assay was performed in microscope chambers so that the samples could be probed directly with an antibody specific for the dystrophin protein. Figure 4 reveals the presence of dystrophin after a 72 hour recovery and 96 hour differentiation period, indicating dystrophin is now being produced within myotubes. Within myotubes that stain positive for dystrophin, it is evident that the protein is distributed throughout (see Figure 4B) These data are in agreement with the western blot analyses performed previously by Bertoni et al. [25]. Altogether, the data from DNA, mRNA and protein analyses show that single stranded oligonucleotides without conjugated chemical moieties at their termini (i.e. Cy3) correct the dystrophin gene in cultures containing an enriched population of myoblasts isolated from dystrophic mice.
Figure 4. Detection of dystrophin with exon specific antibodies. Cultures containing dystrophic myoblasts were seeded in 8 well chambers and were targeted with a 72 hour recovery time after the addition of the either the nonspecific ODN (A) or mdx47NT (B). These cells were then allowed to differentiate for 96 hours at which point they were fixed and incubated with antibody specific for a rabbit polyclonal antibody that recognizes dystrophin and a secondary Alexa fluor labeled antibody (red). The cell nuclei were stained with DAPI, which is shown in blue.
Enhancement of dystrophin correction by knockdown of Msh2
We and others have shown that a transient knockdown of the mismatch repair protein Msh2 in repair proficient cells can increase the overall level of gene repair [26,30]. Msh2 is thought to inhibit the repair reaction through its action as an anti-recombinase [31,32] by precluding the binding of the oligonucleotide. Therefore, by knocking down the levels of this protein, we might predict that the oligonucleotide will bind more stably to the target site with higher efficiency and facilitate oligonucleotide binding. In order to determine whether the repair reaction could be stimulated by knocking down Msh2 in primary cells, we treated our cultures enriched in myoblasts with increasing concentrations of RNAi and quantified the amount of wild-type dystrophin DNA appearing after incubation with the targeting oligonucleotide for 24 hours. The correction frequency was enhanced 2 and 3 fold at the 1 nM and 10 nM concentrations of the RNAi (Figure 5). The efficiency decreases at higher concentrations of RNAi presumably due to the elevated levels of RNAi toxicity often observed in treated cells [33]. The enriched cell cultures used above were purified further to obtain myoblasts. This was accomplished by plating the cells on a collagen coated dish in F10 media. Once confluency was reached, the myoblasts were detached and separated from fibroblasts (etc.); this procedure was carried our repeatedly until the population of cells in the pooled culture was predominantly purified myoblast. The steps in this procedure are illustrated in Figure 6. Panels i and ii display the enriched culture of cells (i) and after differentiation into myotubes (ii); the arrows point to fibroblasts. In panels (iii) and (iv), the purified myoblast culture is illustrated before (i) and after (ii) differentiation into myotubes. Panels (v) and (vi) display cells in dark field after staining with Desmin (red). The fact that the vast majority of cells are stained in panel (vi) indicated that differentiation into myotubes has occurred. Thus, the procedure has enabled the isolation of a purified culture of myoblasts that can be targeted with the oligonucleotide.
Figure 5. Enhancement of Dystrophin repair by knockdown of Msh2. A: Myoblasts derived from mdx5cv mice were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate and significance was deciphered by a student's t-test. Differences were considered significant only when the p < 0.05. B: Knockdown of Msh2 by RNAi. Myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 3 μg of correcting oligonucleotide mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
Figure 6. Dystrophin repair in purified cultures of myoblasts. Purification of the primary cultures of myoblasts. (i) enriched culture of myoblasts. The cultures were derived from limbs of Mdx5cv mice, and population of the myoblasts was enriched using pre-plating technique (see "Materials and Methods); (ii) myotubes in the enriched culture. The enriched cultures were induced to differentiation by culturing in differentiation medium. The arrows show representative fibroblasts; (iii) purified culture of myoblasts. The enriched cultures were subjected to pre-plating repeatedly until fibroblast-like cells were not observed in cultures. The purified cultures were cultivated in F10-based medium; (iv) myotube in the purified culture. The cultures were induced to differentiation by culturing in differentiation medium; (v) and (vi) desmin staining of the purified culture. Cell nuclei were counter stained with DAPI (v; blue). All of the observed cells were positive for desmin staining (vi; red).
The mdx47NT ODN was introduced using jetPEI into a purified myoblast culture at various doses (the N/P ratio was kept at 5) and incubated for 24 hours. Genomic DNA was isolated and RT-PCR performed to analyze the samples for gene repair. We observed a clear dose response by calculating the amount of wild-type DNA emerging in treated samples (Figure 7) as compared to a standard curve (see Methods and Materials). The data suggest that a wild type dystrophin allele is present in at least some of the myoblasts where gene repair should have taken place. While it is not a perfectly sound comparison, the levels of gene repair in the enriched cultures and the purified cultures are approximately equivalent at the 24 hour time point (mdx47NT). Interestingly, the background level from the untreated control samples is higher in the purified myoblasts than in the enriched culture.
Figure 7. Dystrophin correction in the purified cultures of myoblasts. Various amounts of mdx47NT were transfected to purified myoblasts. Total genomic DNA was isolated after 24 hours, and 25 ng of this DNA was subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin.
Since RNAi-directed Msh2 knockdowns had elevated correction efficiencies in myoblast-enriched cultures, we applied this strategy to cultures of purified myoblasts where active conversion was shown (above) to take place. Purified myoblast cultures were treated simultaneously with various doses of the RNAi used in the experiments presented in Figure 5 and either 0.3 μg, 0.6 μg or 0.9 μg of targeting ODN, mdx47NT. The fold change in the evolution of wild type dystrophin DNA was measured after 24 hours and the results are presented in Figure 8A, B and 8C, respectively. No statistically significant difference in correction activity is observed at any RNAi level, although a clear reduction in Msh2 RNA levels is taking place (Figure 9). These data differ from the results reported in Figure 5 suggesting perhaps that enhancement of gene repair by RNAi may not have been occurring in myoblasts.
Figure 8. Effect of Msh2 knockdown on dystrophin correction in purified myoblasts. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and (A) 0.3 μg, (B) 0.6 μg or (C) 0.9 μg of correcting oligonucleotide (mdx47NT), respectively. Total genomic DNA was isolated after 24 hours and 25 ng were subjected to absolute quantification using a FAM labeled probe specific only for wild type dystrophin. The fold change in wild-type dystrophin was determined from three independent experiments assayed in triplicate.
Figure 9. Knockdown of Msh2 by RNAi. Purified myoblasts were co-transfected with various amounts of RNAi specific to knockdown Msh2 and 0.75 μg of correcting oligonucleotide, mdx47NT. Total mRNA was isolated after 6 hours and subjected to RT-PCR.
Successful correction of the dystrophin gene using either the RNA/DNA chimera or the modified single stranded oligonucleotide has been achieved in both dogs and mice by direct injection of synthetic DNA molecules [10-12,25]. The frequency of correction throughout muscle tissues however must be increased to enable therapeutic benefit. Bertoni et al. [25] have demonstrated that when muscle is injected directly with synthetic oligonucleotides, genetic correction was restricted to the dystrophin gene within a population of satellite cells. Since the frequency of gene repair is higher when delivery to cells outside the body takes place in an ex vivo approach. Thus, a more efficient approach for the treatment of DMD might entail using ex vivo delivery with subsequent transplantation back into the host.
To enable this ex vivo strategy, it was essential to optimize the efficiency of ODN delivery and ODN-mediated correction of myoblasts. This was accomplished by transfecting a culture of primary muscle cells enriched in myoblasts with a FAM labeled oligonucleotide and measuring uptake by FACs analysis. We confirmed that the ODN was present in the nucleus by examining the nuclei with confocal imaging (data not shown). Correction of the point mutation in the dystrophin gene was then demonstrated at the level of DNA, RNA and protein. Analysis of the quantity of corrected DNA indicated that the correction frequency remains stable over the course of 96 hours when targeting with either mdx47NT or mdx47T. A significant increase in message containing the portion of exon 10 deleted in the mutant cells was also detected at both 48 and 72 hours after transfection. There was no reduction in the level of repaired RNA over time which suggests that the corrected gene produces stable and inheritable message.
DNA analyses indicate that correction occurs at a 3 fold higher frequency when the nontranscribed strand of the gene is targeted. One hypothesis for strand bias is that the movement of RNA polymerase would interrupt the pairing of the ODN and this would result in lowered correction efficiency [34]. The dystrophin gene, however, is expressed only in differentiated cells and therefore, ODN binding stability should not be disrupted by the movement of the RNA polymerase. Alternatively, the orientation of the gene in terms of the general direction of replication fork movement can influence the targeting frequency in that the lagging strand targets at a higher level than the leading strand [35]. Thus, the strand designated as nontranscribed may also be the lagging strand at the target site.
Previous experiments of Ferrara and Kmiec [36] show that the corrected cells reduce their cell division cycle after introduction of the oligonucleotide because the free DNA ends activate G2 checkpoint proteins. During this time, the uncorrected population is able to divide, which will, in effect, cause the number of corrected cells to be diluted in number. This results in a lower measure of correction efficiencies as division in the uncorrected population is not impaired. In primary myoblasts cultures, however, this does not seem to be the case as the whole population of cells continues to divide with the correction efficiency maintained. This observation suggests that the corrected cells do not suffer a growth disadvantage by this ex vivo protocol.
Duchenne Muscular Dystrophy (DMD) is caused by an absence of the protein dystrophin as indicated by the absence of immunostaining for this protein in most of the muscle fibers. We observed elevated levels of dystrophin in cells that were treated with specific ODNs designed to correct the mutated dystrophin gene and after introduction of the oligonucleotide, myotubes that are expressing a significant amount of dystrophin are detected at 72 hours. The number of corrected cells that contribute dystrophin to the positive myotubes is unknown, but it is interesting to note that each myotube that stained positive had dystrophin evenly distributed throughout. This indicates that the dystrophin produced by the corrected gene can diffuse throughout a small myotube in vitro and would probably diffuse though part of a myofiber in vivo, in a region called the nuclear domain [37]. The long-term goal of the present study is to transplant corrected myoblasts back into mice to restore dystrophin production in muscle fibers. To achieve this goal it was necessary to establish ex vivo targeting conditions wherein the highest level of correction frequency in purified cells is achieved. The observation that Msh2 knockdown enhanced the repair frequency also indicates that the mechanism of gene repair is conserved in primary cells and has, to some degree validated basic studies in established model systems [38]. To our surprise, RNAi knockdown of Msh2 in cultures of purified myoblasts did not lead to an elevation in the repair of the mutant dystrophin gene, regardless of increasing amounts of correcting ODN at the varying doses of RNAi. The overall level of correction in the purified cultures is substantially lower and may be at a basal level where adjuvant treatments designed to stimulate activity do not impact the gene repair reaction. In addition, these results suggest that the stimulation of correction in the enriched cultures was likely taking place in the fibroblasts which contaminate the culture. For unknown reasons, primitive progenitor cells are less amenable to gene repair or at least provide an environment, less conducive for the reaction.
Correction levels vary among cell types with certain mutant targets being repair at levels approaching 1–5%. Yet, the consensus view is that progenitor cells, at any step along the differentiation pathway, do not enable this reaction to take place efficiently [39-41]. A series of natural barriers seems to exist in these cell types including lower transfection efficiencies and dosage-dependent toxicity. Increasing the amount of ODN introduced into the cell usually results in a gradual increase in side effects [39]. Progenitor cells are usually more sensitive to the transfection agent [42] and strand bias for targeting can also vary widely based in gene types [43]. Murphy et al. [39] found a mismatch hierarchy that contributed to the various levels of gene correction; A:G > A:A > A:C. If this is true, then the capacity to target progenitor cells will be hampered. Such a hierarchy has not been observed in somatic cell targeting where correction efficiencies are generally higher.
Thus, progenitor cells will present unique challenges that will require exogenous manipulation to elevate frequency of gene repair. While MSH2 knockdown appears not to be a sufficient protocol to meet this goal in myoblasts, it does not mean that other progenitor lineages will not response positively. In fact, Dekker et al. [41] have already shown that MSH2 knockdown does increase the frequency of gene repair in mouse ES cells. More recently, Aarts et al. [30] provided support and confirmed these observation and Maguire and Kmiec [26] extended this protocol successfully into mouse embryonic fibroblasts. Morozov and Wowrousek [44] followed among the same theme with observations that inhibition of Rad51/54 and Ku70/86 led to an increase in gene correction efficiency. These types of cellular manipulations are certainly feasible and may be required for the successful application of gene repair in primary or progenitor cells.
Cells and oligonucleotides
Primary myoblasts derived from the mdx5cv mice were maintained in DMEM supplemented with either 10% or 20% FBS. When the cells were induced to differentiate, DMEM containing 2% horse serum (DM) was added to the cells for at least 96 hours. The mutation of the parental plasmid pEGFP N3 to create the mutant pEGFP N3 (rep) has been described previously [38]. Targeting oligonucleotides, eGFP3S/47NT, mdx47NT, mdx47T, and mdx47NT-FAM were obtained either from Sigma Genosys (St. Louis, MO) or IDT (Coralville, IA) where they were synthesized and purified by HPLC before distribution. All modified single-stranded oligonucleotides were resuspended to a concentration of 200 μM and aliquots were stored at -20°C. The mdx5cv mice were obtained from the Jackson Laboratory.
Isolation of myoblasts from newborn mdx5cv mice
The isolation procedure for mouse myoblasts and subsequent pre-plating methods have been described previously [27,28]. This work was authorized by the Laval University Animal Care Committee and experiments were conducted in accordance with guidelines set forth by the Canadian Council of Animal Care. Briefly, limbs of 1 to 3 days old male mdx5cv mice were isolated and muscle was pulled from the bone and cartilage using sterile forceps. The muscle was treated for 1 hour with Collagenase (Sigma, St. Louis, MO) and Dispase II (Roche, Indianapolis, IN) to degrade collagen and to disrupt cell contacts. The cells were plated for 2 hours to allow fibroblasts, which rapidly adhere, to attach to the petri dish (PP2). The supernatant, containing a mixture of myoblasts and fibroblasts, was removed and the cells are passaged again to another petri dish (PP3). After 24 hours the cells were again passaged (PP4) and subsequently transferred to another culture dish (PP5). Purified myoblasts were expanded and used in gene repair experiments.
Cells were aliquoted into 4 wells of an 8 well chamber at a density of 1 × 105 cells, and allowed to attach overnight. The cells were fixed using 100% ethanol for 5 minutes at -20°C, permeabilized with 0.1% Triton X-100, and then incubated with either desmin (Sigma, St. Louis, MO) specific or dystrophin specific antibodies raised in rabbit (Abcam, Cambridge, MA 1:1,000) at room temperature (RT) for 1 hour or overnight at 4°C. After washing the cells, a secondary goat α-rabbit antibody conjugated to a Cy3 fluorophore (desmin) (Zymed Laboratories, San Francisco, CA) or an Alexa 546-coupled goat anti-rabbit antibody (Molecular probes 1:1500) was added to the wells and this mixture was incubated with agitation for 1 hour at room temperature. After four 15 minute washes, the wells were dried and mounting media containing DAPI was added to each well (Molecular Probes, Eugene, OR). The cells were then visualized using a Zeiss™ inverted 100 M Axioscope equipped with a Zeiss™ 510 LSM confocal microscope with a coherent krypton argon laser (30 mW) containing 488 nm or 568 nm and 742 nm excitation lines.
Gene repair of dystrophin in purified myoblasts
Cells were plated in 6-well culture dishes at a density of 1 × 105 with media containing 20% FBS. jetPEI was used to make oligonucleotide complexes with 3 μg of ODN at an optimal N/P ratio (nitrogen/phosphate ratio) of 5. This reagent was added to 1 ml of media and the cells were incubated for various amounts of time at which point differentiation media (DM) was added; the cells were differentiated for 96 hours, then harvested for FACS analysis and genomic DNA isolation. For mRNA analyses, the cells were collected at least four days after the addition of DM containing 2% horse serum.
Knockdown of Msh2
RNAi, designed to knock down Msh2, was purchased from Ambion (Ambion, Austin, Texas); siRNA (ID# 155431) is designed to anneal to the mouse MSH2 RNA at exon 3 NM_008628. During the targeting experiments, the RNAi was mixed with the correcting oligonucleotides complexed with jetPEI and added to the cells as described above.
Co-transfection ssODNs and Msh2 RNAi
Cells were plated in 6-well plates at a density of 1 × 105 cells/well with growth media (DMEM supplemented with 20% FBS, penicillin and streptomycin). Twenty four hours after plating, cells were co-transfected 3 μg of mdx/47NT and RNAi specific to knockdown Msh2 (0–200 nM). Seventy two hours after transfection, the growth media were replaced by differentiation media (DMEM supplemented with 2% horse serum, penicillin and streptomycin), and the cells were maintained in differentiation media for 96 h. Total RNA was isolated from the cells, and reverse transcription and quantitative PCR were conducted as described in "RNA isolation and, RT PCR and quantitative PCR.
Msh2 RNAi
Cells were plated in 6-well plates at a density of 1 × 105 cells/well with growth media. Twenty four hours after plating, cells were co-transfected 3 μg of mdx47NT and RNAi specific to knockdown Msh2 (1–100 nM). Total RNA was isolated 3 or 6 h after transfection Methods of reverse transcription and quantitative PCR were same as "RNA isolation and, RT PCR and quantitative PCR", except for primers: musMSH2/257F 5' agaccctgcagagtgttgtgctta 3'; musMSH2/1010R 5' tgagagccagtggtgtcttcaaca 3' or mouse GAPDH4FwQ 5' tgtctcctgcgacttcaacagc 3'; GAPDH5RevQ 5' atgtaggccatgaggtccacca 3'.
RNA isolation, RT PCR and quantitative PCR
After differentiation of the myoblasts, the cells were harvested by trypsinization and collected by centrifugation at 1,500 rpm for 7 minutes. After washing with 1× PBS, the RNA was extracted using a Qiagen RNA extraction kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Three hundred nanograms of RNA was subjected to reverse transcription to generate cDNA using a gene specific primer Rev-ex12 5' cag gtc caa agg gct ctt cc 3' or primer oligo dT using the first strand synthesis kit (Invitrogen, Carlsbad, CA). Five microliters was added to a 20 μL mixture of 2× Sybrgreen Master Mix (Applied Biosystems, Foster City, CA) with 2.5 μM of each primer:
Forw-ex 9 5' gtt atg cct tca cag gct gc 3'; Rev-ex10 in 5'cacttc ttc aac atc att tg3' or GAPDH4FwQ 5'tgtctcctgcgacttcaacagc3'; GAPDH5RevQ 5'atgtaggccatgaggtccacca 3' CKM F1 5' GCAACACCCACAACAAGTTCA 3' and CKM R1 5' TGTCTCCTTATCGCGAAGCTT 3'. Samples were added to each well of a 96 well plate in duplicate for each primer set. Dystrophin was amplified using the cDNA generated by the gene specific primer and GAPDH or CKM from cDNA generated using oligo dT. QPCR was run for 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes, 40 repetitions at 95°C for 15 seconds and 40 repetitions at 59°C for 1 minute with data collection at step 1 of stage 3. Upon completion of PCR, the threshold and baseline values were set to measure Ct values at the beginning of the exponential amplification. Ct values from 3 experiments were averaged per sample and subjected to a student's t-test and ANOVA to determine significance.
DNA isolation and absolute quantification by PCR
Cells were harvested and collected by centrifugation at 1500 rpm for 7 minutes and washed twice with 1× PBS or lysed directly in a tissue culture dish after washing with 1× PBS. DNA was precipitated using the genomic wizard DNA extraction kit as instructed by the manufacturer (Promega, Madison WI). Twenty-five nanograms of genomic DNA was added to a total of 7 μL of water and this was mixed with 13 μL of a 2× PCR master mix, 50 nM of a 6FAM labeled probe specific to bind to wild-type dystrophin 5'FAMcac aag gag aga ttt3', 900 nM of forward primer mdx5cvasF 5' ttt ctg ccg agg ata cat tgc3' and 900 nM of reverse primer ex 10 out 5' ctc atg agc atg aaa ctg ttc 3'. Each experimental protocol was run in triplicate. A standard curve was generated using 50 ng, 25 ng, 12.5 ng, 6.25 ng, and 3.13 ng of genomic DNA isolated from C2C12 cells, which are wild-type for dystrophin. As a separate control, reaction mixtures containing 25 ng of genomic DNA from mdx5cv were used to determine the background of wild-type DNA in each experiment. PCR conditions were: 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The data were collected at stage 3 of step 1. Threshold and baseline levels were set to measure the Ct values at the lowest level of exponential amplification and the average mean was determined from each experiment. Statistical significance was determined by a student's t-test and was considered significant only if p was less than 0.05. Following the PCR amplification the products were purified using Qiagen's QIAquick PCR purification kit according to the instructions and submitted for sequencing using the mdx5cvasF primer.
Primary mouse myoblast purification
The enriched culture obtained from Tremblay lab was plated on a collagen-coated culture dish with F10 nutrient mixture (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) FBS, 2.5 ng/ml bFGF (Promega, Madison, WI), 100 U/ml penicillin and 100 mg/ml streptomycin. When the culture reached confluency, cells were detached from the dish without using trypsin. To accomplish this, the dish was washed with PBS after aspiration of medium, incubated with a small amount of PBS at room temperature for 5 min, and hit firmly sideways. Predominant cell type in the detached cells was myoblast due to the fact that myoblasts are likely to detach from collagen-coated dish than fibroblasts are. The detached cells were collected with F10 nutrient mixture, plated on a new collagen-coated dish, and expanded. The above procedures were repeated until no fibroblasts were observed in the culture. Upon completion of purification, medium was switched F10 growth medium to F10/DMEM (1:1) supplemented with 10% (v/v) FBS, 2.5 ng/ml bFGF (Promega, Madison, WI), 100 U/ml penicillin and 100 mg/ml streptomycin.
Desmin staining
Cells were plated on 2-well chambers at a density of 8 × 104 cells/well and incubated at 37°C overnight. The cells were fixed using ethanol for 5 minutes at -20°C, permeabilized using 0.3% (v/v) TritonX for 10 minutes at room temperature, blocked using 5% (v/v) heat-inactivated goat serum for 30 minutes at room temperature, and then incubated with desmin specific antibody (Sigma, St. Louis, MO; dilution, 1:50) for 1 hour at room temperature. After washing the cells, a secondary goat a-rabbit antibody conjugated to Cy3 (Zymed laboratories, San Francisco, CA; dilution, 1:750) was added to the wells and this incubated with agitation for 1 hour at room temperature. After four 15 minute washes, the wells were briefly dried and mounting media containing DAPI was added to each well (Molecular Probes, Eugene, OR).
Oligo dose experiment
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. jetPEI was used to make oligonucletide complexes with various amounts of mdx47NT at N/P (nitrogen/phosphate) ratio of 5. This reagent was added to 1 ml of media and the cells were incubated. Genomic DNA was isolated 24 hours after transfection using masterpure DNA purification kit (Epicentre, Madison, WI). TaqMan® probe-based real time PCR was performed to quantify wild type dystrophin. Probe specific for wild type dystrophin (5'-FAMcac aag gag aga ttt-3') were used with the primer pair: forward primer, 5'-ttt ctg ccg agg ata cat tgc-3'; reverse primer, 5'-ctc atg agc atg aaa ctg ttc-3'. Real time PCR was performed with 25 ng genomic DNA, 900 nM both forward and reverse primer, and 250 nM probe in each 20 ml reaction. A standard curve was generated using 1, 0.5, 0.25, 0.125, 0.0625 ng of genomic DNA isolated from C2C12 cells, which have wild type dystrophin gene. PCR conditions were: 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each experimental protocol was run in triplicate.
Co-transfection ssODNs and Msh2 RNAi
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. Cells were co-transfected 0.75 mg of mdx47NT and siRNA for Msh2 knockdown (0–100 nM) using jetPEI. Twenty four hours after transfection, genomic DNA was isolated, and TaqMan® probe-based real time PCR was performed to quantify wild type dystrophin as described above ("Oligo dose experiment")
Msh2 RNAi
Purified myoblasts were plated in 6-well plates at a density of 1.5 × 105 cells/well with F10/DMEM growth medium 24 hours before transfection. Cells were co-transfected 0.75 mg of mdx47NT and siRNA for Msh2 knockdown (0–100 nM) using jetPEI. Total RNA was isolated 6 hours after transfection. Two hundred nanograms of RNA was subjected to reverse transcription to generate cDNA using the first strand synthesis kit (Invitrogen). Five microliters was added to a 20 ml mixture of 2× Sybergreen master mix (Applied Biosystems, Foster City, CA) with 2.5 mM of each primer: musMSH2/257F 5' agaccctgcagagtgttgtgctta 3'; musMSH2/1010R 5' tgagagccagtggtgtcttcaaca 3' or mouse GAPDH4FwQ 5' tgtctcctgcgacttcaacagc 3'; GAPDH5RevQ 5' atgtaggccatgaggtccacca 3'. Samples were added to each well of a 96 well plate in duplicate for each primer set. QPCR was run for 1 repetition at 50°C for 2 minutes, 1 repetition at 95°C for 10 minutes, 40 repetitions at 95°C for 15 seconds and 40 repetitions at 59°C for 1 minute with data collection at step 1 of stage 3. Upon completion of PCR, the threshold and baseline values were set to measure Ct values at the beginning of the exponential amplification. Ct values from 3 experiments were averaged per sample.
ODN: Single-stranded oligonucleotides; DMD: Duchenne Muscular Dystrophy.
Authors' contributions
KM carried out the experiments in enriched myoblasts along with the initial RNAi work; TZ purified the myoblasts and carried out the experiments in the purified cells. KM and HPO in conjunction with EK conceived the study; HPO and EK drafted the manuscript. All authors read and approved the final manuscript.
We would like to thank Dr. J. Tremblay's lab for providing the enriched myoblasts and for critical reading of the manuscript (Unité de recherche en Génétique Humaine, Centre de Recherche du Centre Hospitalier de Université Laval, CHUL du CHUQ, Ste-Foy, QC, Canada). This project was supported by NIH R21 NS053608.
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Vanilla and cinnamon add sweetness while chipotle chile pepper and ginger bring an unexpected heat to these grilled fruit skewers. For variety, add sliced star fruit to this jazzed up dessert.
1/2 cup packed brown sugar
1/4 cup rum
2 teaspoons McCormick® Pure Vanilla Extract
1/2 teaspoon McCormick Gourmet™ Cinnamon, Ground Saigon
1/2 teaspoon McCormick Gourmet™ Ginger, Ground
1/4 teaspoon McCormick Gourmet™ Chile Pepper, Chipotle
1 large pineapple, peeled, cored, cut into 2x1-inch pieces
2 ripe bananas, peeled, cut into 2x1-inch pieces
Stir first 6 ingredients in small bowl until sugar dissolves. Set aside.
Thread fruit alternately onto 6 double skewers. Brush with spice mixture. Let stand 5 minutes.
Grill or broil fruit skewers on medium heat 10 minutes or until golden brown, basting with spice mixture and turning occasionally. Serve with vanilla ice cream, if desired.
• If using wooden skewers, soak in water for at least 30 minutes.
• To prevent pieces of fruit from spinning around as you turn kabobs, thread fruit onto two parallel skewers for each kabob.
(Amount per serving)


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Transit of Venus Videos
March 22, 2012
A four-minute trailer summarizes the history and significance of the transit of Venus while preparing for June 5-6, 2012, spectacle. Story traces its value from early expeditions seeking to measure the size of the solar system, to similarities with transits around distant stars being detected by the Kepler spacecraft. credit: NASA
Topics: Environment, Astrological aspects, Astrology, Astronomy, Technology Internet, Kepler Mission, Transit of Venus, Astronomical transit, Johannes Kepler, Astrometry, Venus, Spacecraft, Venus Videos


text:
Ranchita evacuation order to be lifted
As of Friday morning the Wilson Fire is 11,691 acres and 75% contained and the Stewart Fire is 10,630 acres and 90% contained.
Evacuations orders for Ranchita and San Felipe will be lifted as of 9 a.m., according to Cal Fire.
NORTH COUNTY Firefighters were racing against worsening weather conditions Thursday afternoon as they continued gaining ground on two major fires burning near Ranchita, a Cal Fire official said.
With the progress, state Route 78 reopened to traffic about 1 p.m., but evacuated Ranchita and San Felipe residents still were not allowed to return home.
The Wilson fire was 12,000 acres and 75 percent contained as of Thursday evening. A little further south, the Stewart fire was 7,100 acres and 65 percent contained, Cal Fire said.
Both were ignited by lightning strikes in the desert Monday near Scissors Crossing northeast of Julian and scorched their way toward Ranchita, prompting evacuation orders Tuesday night for the community of about 400 people.
The two fires are the largest of five started by lightning in the northeastern part of the county this week, and the only ones not fully contained. No structures have been damaged.
"We're continuing to make progress," Cal Fire Capt. Mike Mohler said Thursday. "But the weather is changing. There are thundershowers in the area. I don't see any lightning yet. If we're going to get lightning, I hope we get some moisture, too."
The National Weather Service issued a hazardous weather outlook for possible flash floods and thunderstorms.
Mohler said fire authorities were considering earlier in the day whether they could lift the evacuation order, with the Wilson fire half a mile from the nearest homes along Montezuma Valley Road.
"We have a large containment barrier between the fire and homes," Mohler said.
But with the turn in the weather, the order remained in place.
Gary and Jeanine Christensen of San Felipe sheltered Tuesday and Wednesday nights at the Warner Springs Middle/High School, where the Red Cross set up an evacuation center in the gym.
The couple had the county Department of Animal Services help them evacuate their three goats, four chickens, four cats, two dogs and a rabbit before heading to the shelter.
"I just don't take chances. It's not worth it," said Jeanine Christensen, 45. She said they were worried that if they waited until the fire got any closer, they wouldn't be able to get out the dirt road leading to their 1½-acre property.
Mohler said winds from the west on Wednesday afternoon pushed flames away from Ranchita. That allowed fire crews to set controlled burns, charring grass and brush before the wildfire could reach it.
"We probably would not have done that if the winds were not in our favor," Mohler said. "When we had a successful line, a black line of hundreds of acres, that really brought our containment up.
"From now on, we want to get ground crews in there and strengthen the line."
With lightning strikes, aircraft have to be grounded and crews on foot leave the fire lines for safer ground, Cal Fire Battalion Chief Mike Smith said. He said showers also bring a danger of flash floods and road damage, which also hamper firefighting efforts.