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Patent US20020165574 - Emboli protection devices and related methods of use - Google Patents
An evacuation sheath assembly and method of treating occluded vessels which reduces the risk of distal embolization during vascular interventions is provided. The evacuation sheath assembly includes an elongated tube defining an evacuation lumen having proximal and distal ends. A proximal sealing surface...http://www.google.com/patents/US20020165574?utm_source=gb-gplus-sharePatent US20020165574 - Emboli protection devices and related methods of use
Publication number US20020165574 A1
Also published as US7374560, US20080243171
Publication number 09940986, 940986, US 2002/0165574 A1, US 2002/165574 A1, US 20020165574 A1, US 20020165574A1, US 2002165574 A1, US 2002165574A1, US-A1-20020165574, US-A1-2002165574, US2002/0165574A1, US2002/165574A1, US20020165574 A1, US20020165574A1, US2002165574 A1, US2002165574A1
Inventors Thomas Ressemann, Steven Hackett, Andrew Dusbabek, Scott Olson, Peter Keith, Dennis Wahr
Original Assignee Velocimed.
Patent Citations (99), Referenced by (85), Classifications (27), Legal Events (5)
US 20020165574 A1
This is a continuation-in-part of application Ser. No. 09/845,162, filed May 1, 2001, which is incorporated herein by reference.
Several systems to prevent emboli being released into the bloodstream during such procedures have been tried. One system uses a balloon to totally occlude the artery distal (downstream) to the area of blockage to be treated. In this system, a guidewire with a balloon is introduced into the narrowed or occluded area, and passes through the narrowed or occluded area to a position downstream of the blockage. The balloon is inflated, the blockage is reduced or removed, and then the blood proximal to the balloon is withdrawn from the blood vessel to remove any particles or emboli which decrease in emboli related complications in patients undergoing such treatments, the event rate remains significant. One particular problem with this system is passing the guidewire and balloon through the narrowed or occluded area prior to occlusion with the balloon, creating the risk that emboli will be produced as the balloon passes through the blockage. Thus, any particulate or plaque disturbed during this passage which forms emboli prior to inflation of the balloon is free to flow through the vascular system, increasing the risk for infarction or ischemia. Also, any debris or particulate matter which gathers around the edges of the balloon may slip downstream during deflation and retrieval of the balloon. In addition, this system requires that blood flow be totally occluded in the vessel for relatively prolonged intervals that may induce adverse cardiac events. Although this may not be a problem clinically, many patients perceive the occlusion of blood flow for this period of time as problematic.
[0047]FIG. 1A is a cross-sectional side view of a partial length evacuation sheath according to one embodiment of the present invention;
[0048]FIG. 1B is a cross-sectional view of the partial length evacuation sheath taken along line 1B-1B of FIG. 1A;
[0049]FIG. 1C is a cross-sectional side view of an alternative embodiment of a partial length evacuation sheath according to one embodiment of the present invention;
[0050]FIG. 1D is a cross-sectional view of the partial length evacuation sheath taken along line 1D-1D of FIG. 1C;
[0051]FIG. 2A is a cross-sectional side view of an expandable evacuation sheath, shown in an unexpanded state, according to another embodiment of the present invention;
[0052]FIG. 2B is a cross-sectional view of the unexpanded expandable evacuation sheath taken along line 2B-2B of FIG. 2A;
[0053]FIG. 2C is a cross-sectional side view of the expandable evacuation sheath of FIG. 2A in an expanded state;
[0054]FIG. 2D is a cross-sectional view of the expanded expandable evacuation sheath taken along line 2D-2D of FIG. 2C;
[0055]FIG. 2E is a cross-sectional view of the expanded evacuation sheath taken a long line 2E-2E of FIG. 2C.
[0056]FIG. 3A is cross-sectional side view of a full-length evacuation sheath according to another embodiment of the present invention;.
[0057]FIG. 3B is cross-sectional view of the full-length evacuation sheath taken along line 3B-3B of FIG. 3A;
[0058]FIG. 4A is cross-sectional side view of a guiding catheter/evacuation sheath combination according to yet another embodiment of the present invention;
[0059]FIG. 4B is cross-sectional view of the guiding catheter/evacuation sheath combination taken along line 4B-4B of FIG. 4A;
[0060]FIG. 5A is cross-sectional view of the partial evacuation sheath of FIGS. 1A and 1B deployed within a vessel;
[0061]FIG. 5B is cross-sectional view of the expandable evacuation sheath of FIGS. 2A-2D deployed within a vessel;
[0062]FIG. 5C is cross-sectional view of the full-length evacuation sheath of FIGS. 3A and 3B deployed within a vessel;
[0063]FIG. 5D is cross-sectional view of the guiding catheter/evacuation sheath combination of FIGS. 4A and 4B deployed within a vessel;
[0068]FIG. 10A is a cross-sectional side view of another embodiment of an evacuation sheath assembly enclosed in a delivery sheath and being delivered through a guiding catheter;
[0069]FIG. 10B is a cross-sectional side view of a braided sheath forming an evacuation head of the evacuation sheath assembly of FIG. 10A in an unexpanded state with the delivery sheath removed;
[0070]FIG. 10C is a cross-sectional side view of the braided sheath of FIG. 10B in the expanded state; and
[0071]FIG. 10D is cross-sectional view of the guiding/evacuation lumen of the evacuation sheath assembly of FIGS. 10A-10C deployed within a blood vessel.
[0072]FIG. 11A is a cross-sectional side view of a partial length evacuation sheath according to one embodiment of the present invention;
[0073]FIG. 11B is a cross-sectional view of the partial length evacuation sheath taken along line A-A of FIG. 11A;
[0074]FIG. 11C is a cross-sectional side view of a partial length evacuation sheath according to one embodiment of the present invention;
[0075]FIG. 11D is a cross-sectional view of the partial length evacuation sheath taken along line A-A of FIG. 11C;
[0076]FIG. 11E is a cross-sectional side view of a partial length obturator according to one embodiment of the present invention;
[0077]FIG. 11F is a cross-sectional view of the partial length obturator taken along line A-A of FIG. 11E;
[0078]FIG. 11G is a cross-sectional side view of the partial length obturator located within a partial length evacuation sheath;
[0079]FIG. 11H is a cross-sectional side view of partial length balloon obturator according to one embodiment of the present invention;
[0080]FIG. 11I is a cross-sectional view of the partial length balloon obturator taken along line A-A of FIG. 11H;
[0081]FIG. 11J is a cross-sectional view of the partial length balloon obturator taken along line B-B of FIG. 11H;
[0082]FIG. 11K is a cross-sectional side view of the partial length balloon obturator located within a partial length evacuation sheath;
[0083]FIG. 12A is a cross-sectional side view of an infusion catheter according to one embodiment of the present invention;
[0084]FIG. 12B is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12A;
[0085]FIG. 12C is a cross-sectional view of the infusion catheter taken along line B-B of FIG. 12A;
[0086]FIG. 12D is a cross-sectional side view of an infusion catheter according to one embodiment of the present invention;
[0087]FIG. 12E is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12D;
[0088]FIG. 12F is a cross-sectional view of the infusion catheter taken along line B-B of FIG. 12D;
[0089]FIG. 12G is a cross-sectional side view of an alternative infusion catheter according to one embodiment of the present invention;
[0090]FIG. 12H is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12G;
[0091]FIG. 12I is a cross-sectional side view of another infusion catheter according to one embodiment of the present invention;
[0092]FIG. 12J is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12I;
[0093]FIG. 12K is a cross-sectional side view of an infusion catheter according to one embodiment of the present invention;
[0094]FIG. 12L is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12K;
[0095]FIG. 12M is a cross-sectional side view of an infusion catheter according to one embodiment of the present invention;
[0096]FIG. 12N is a cross-sectional view of the infusion catheter taken along line A-A of FIG. 12M;
[0097]FIG. 13 is a cross-sectional view of the partial length evacuation sheath of FIGS. 11A and 11B deployed in a blood vessel according to a further aspect of the present invention;
[0098]FIG. 14 is a cross-sectional view of the partial length evacuation sheath of FIGS. 11A and 11B and the over the wire infusion sheath of FIGS. 12K and 12L deployed in a blood vessel according to a further aspect of the present invention; and
[0099]FIG. 15 is a cross-sectional view of a heart with a coronary sinus partially occluded by an occlusion catheter according to one aspect of the present invention.
[0112]FIG. 11C is an alternative embodiment of an evacuation sheath assembly according to the present invention. This embodiment is similar to that described in connection with FIGS. 11A and 11B, except that the distal tip of the evacuation head 132 is cut perpendicular to the axis of the evacuation lumen 140 and proximate to the distal sealing balloon 136. The perpendicular cut is useful when the anatomy is such that an angled distal end would contact the vessel wall in a way which limits fluid flow through evacuation lumen 140. FIG. 11D is a cross-sectional view of the assembly shown in FIG. 11C.
[0118]FIG. 11K shows the balloon obturator assembly 1000 positioned in a previously described evacuation sheath assembly 100 as it would be used in the blood vessel 150. The balloon 1090 is inflated against the evacuation lumen 140 to secure the balloon obturator assembly 1000 to the evacuation sheath assembly 100 during use. In this position, the balloon obturator assembly 1000 provides a gradual diameter transition from the distal guide wire lumen 1030 b, to the distal end of the evacuation sheath assembly 100. The balloon obturator assembly 1000 is designed to be deflated and removed from the blood vessel 150 after the evacuation sheath assembly 100 has been properly located in the blood vessel.
Fluid continues to be injected into the balloons until proximal balloon 234 creates a seal with the lumen of guide catheter 260 and until distal balloon 236 creates a seal with blood vessel 250. After the proper seals are established, the stenosis 280 is treated and any embolic debris 297 is removed via retrograde flow 295 (FIGS. 7C-7H), as previously described with respect to FIGS. 6C-6H. After treatment, evacuation head 232, including proximal and distal sealing balloons 234, 236, is deflated and then removed from blood vessel 250 (FIG. 71).
The balloon 193a of the stent delivery catheter 193 is then re-inflated within the stent site 194. As shown in FIG. 13, while intervention balloon 193 a is inflated, a vacuum is applied to the guide catheter 160, and the proximal and distal sealing balloons 134, 136 are deflated, allowing blood from the aorta 191 to flow into the vessel 150 and past the end of evacuation sheath assembly 100. Preferably for this method, the guide catheter 160 and evacuation head 132 are sized such that retrograde flow of blood entering into the lumen of the guide catheter 160 enters primarily via the lumen 140 of the evacuation head 132, and not directly into the distal end of the guide catheter 160. This blood flow 191 is then caught by the flow reversal caused by the vacuum applied to the guide catheter 160, and reverses into the evacuation sheath.
[0220]FIGS. 12G and 12H show a further alternative infusion catheter assembly 700. The infusion catheter assembly 700 includes an infusion/guide wire lumen 740 having a proximal end 740 a and a distal end 740 b. Infusion/guide wire lumen 740 is contained within an infusion catheter shaft 770 having a proximal end 755 a and a distal end 755 b, which allows for the passage of a guide wire. The infusion/guide wire lumen 740 is also designed to allow for fluid flow within the infusion/guide wire lumen 740. This fluid flow may occur regardless of whether a guide wire is within the infusion/guide wire lumen 740. The infusion catheter shaft 770 is preferably made of polyethylene. Alternatively, the infusion catheter shaft 770 may be constructed of other polymers, polymer and metal composites, or other suitable biocompatible materials.
[0224]FIGS. 12M and 12N show another infusion catheter assembly 800. The infusion catheter assembly 800 includes a proximal infusion lumen 840 a contained within a proximal shaft 870. The proximal infusion lumen 840 a is designed to allow for fluid flow between a fluid source (not shown) and a distal infusion lumen 840 b. The infusion catheter assembly 800 preferably does not contain a guide wire lumen, and is delivered within the vessel lumen without being tracked over the indwelling guide wire.
Additionally, a stiffness transition member 880 is attached to a distal end 870b of the proximal shaft 870. The stiffness transition member 880, preferably made of stainless steel or alternatively of other metals or composites, extends within the infusion lumen 840 b of the distal shaft 890. The stiffness transition member 880 preferably has a stiffness that decreases along its length from its proximal end 880 a to its distal end 880 b. The decrease in stiffness is attributed to a reduction in the cross sectional area of the member 880. The stiffness transition member 880 is preferably sealingly bonded to the distal end 890 b of the distal shaft 890 and may extend distally beyond the bond. The portion of the stiffness transition member 880 that extends beyond the distal shaft 890 is surrounded by and fixed to a spring coil 815. The spring coil 815 is preferably made of platinum or another metal of a density suitable for visualization by fluoroscopy. The stiffness transition member 880 and the spring coil 815 assembly can be bent into a predetermined shape. The predetermined shape is designed to help steer the assembly 800 through turns in the blood vessel, without the need for tracking over the indwelling guide wire. Alternately, there may be no distally extending spring coil 815. Preferably, the proximal shaft 870 contains proximal shaft markers 820, providing a structure for determining when the catheter has been advanced to a location just proximal to the distal tip of the evacuation sheath.
US6837881 * Jul 2, 2003 Jan 4, 2005 Coaxia, Inc. Devices and methods for preventing distal embolization using flow reversal by partial occlusion of the brachiocephalic artery
US6878128 * Apr 6, 2004 Apr 12, 2005 Kerberos Proximal Solutions Fluid exchange system for controlled and localized irrigation and aspiration
US6905505 * Apr 12, 2002 Jun 14, 2005 Kensey Nash Corporation System and method of use for agent delivery and revascularizing of grafts and vessels
US20020029031 * Jan 25, 2001 Mar 7, 2002 Bagaoisan Celso J. Methods for emboli containment
US20040054347 * Apr 15, 2003 Mar 18, 2004 Gholam-Reza Zadno-Azizi Method for containing and removing occlusions in the carotid arteries
US20040069549 * Oct 7, 2003 Apr 15, 2004 Yamaha Hatsudoki Kabushiki Kaisha Electric vehicle
WO2012156924A1 * May 16, 2012 Nov 22, 2012 Cardioflow Ltd. Vascular occlusion and aspiration device
U.S. Classification 606/194, 604/101.01, 604/509
International Classification A61B17/12, A61M25/00, A61F2/06, A61F2/82, A61B17/22, A61B17/00, A61B19/00, A61F2/84
Cooperative Classification A61B90/39, A61B2217/005, A61B2017/22082, A61B2017/22084, A61B2017/22079, A61B2217/007, A61B17/12109, A61F2/95, A61B2017/22067, A61B17/22, A61B17/12136, A61B17/12045, A61B2017/12127


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Best Price on Hotel Les Embruns in Le Touquet + Reviews
Hotel Les Embruns89 Rue De Paris, Le Touquet City Center, Le Touquet, France - View on mapVery good8.0Based on248reviewsHotel Les EmbrunsSee more photosSee more photosBathroomSee more photos8.0 Very good guest ratingSee all reviews"the cleanliness and menu of the breakfast bar, also being ab..."CharlesUnited KingdomEntranceSee more photos8.9 Excellent locationSee mapSee all 44 photos8.9LocationFeatures you'll loveClose to public transportationFree Wi-Fi in all roomsGardenCar park
See photos and detailsTriple Room14 sq.m.1 single bed and 1 double b...Enter dates to see pricesSee photos and detailsDouble Hot DealEnter dates to see pricesSee photos and detailsDouble Hot DealEnter dates to see pricesSee photos and detailsSuperior Double Room15 sq.m.1 king bedEnter dates to see pricesSee photos and detailsTwin Hot DealEnter dates to see pricesSee photos and detailsDouble Hot DealEnter dates to see pricesSee photos and detailsDouble Room Single Occupancy10 sq.m.1 double bedEnter dates to see pricesSee photos and detailsTwin Room15 sq.m.2 single bedsEnter dates to see pricesSee photos and detailsDouble Hot DealEnter dates to see pricesSee photos and detailsSingle Hot DealEnter dates to see pricesSee photos and detailsQuadruple Room15 sq.m.2 single beds and 1 double ...Enter dates to see pricesSee photos and detailsStandard Double Room1 double bedEnter dates to see prices
More about Hotel Les EmbrunsAn overviewA 3-minute walk from the beach and from the city centre, this hotel proposes individually styled rooms with free Wi-Fi. It is a 5-minute drive from Le Touquet Congress Centre and features a private garden.
The property is 38 km from Boulogne sur Mer.Show moreWhat they offerInternet accessFree Wi-Fi in all roomsWi-Fi in public areas Getting aroundCar park Things to do, ways to relaxGarden Services and conveniencesSmoking area For the kidsFamily room AccessPets allowed Show lessGuest policiesExtra beds are dependent on the room you choose. Please check the individual room capacity for more details.When booking more than 5 rooms, different policies and additional supplements may apply.Show moreAnnouncementsPlease note that pets are allowed for an additional fee of EUR 18 per pet per day.

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Rain plays spoilsport as Mumbai schools, colleges tweak Yoga Day plans | mumbai | Hindustan Times
Rain plays spoilsport as Mumbai schools, colleges tweak Yoga Day plans
Given the incessant rains lashing Mumbai from Friday, few city schools and colleges will be able to keep their date with the rest of the world and celebrate Yoga Day on Sunday.
mumbai Updated: Jun 20, 2015 21:57 IST
While some educational institutions rescheduled the event to either Monday or Tuesday, others held the Yoga Day-related programmes on Saturday itself. Some colleges even had to cancel their celebrations on Saturday. “We preponed our celebrations to Saturday as heavy showers have been forecast for Sunday,” said Anjali Bowen, principal of Ryan International School, CBSE, Kandivli.
Schools that did not have classes on Saturday have moved the celebrations to Monday or Tuesday. “We have not been able to plan the day as the school was closed on Friday and Saturday,” said Sister Maria, principal, St Teresa’s Convent High School, Santacruz. Activity School in Peddar Road, Balmohan Vidya Mandir in Dadar and several others plan to celebrate Yoga Day on Monday.
Some schools though will brave the rains and observe the event on Sunday. “We planned to postpone the event. But we received misleading messages from the education department that it was compulsory to observe the day on Sunday,” said Rajesh Pandya, senior teacher, Fatimadevi English School, Malad. “It will be a tough as we do not have a huge campus or an indoor hall,” he added.
Some colleges too have decided to keep the date with the rest of the world. Thakur Vidya Mandir School and Junior College, Kandivli, has asked students to dress in. “We do not want to miss out on celebrating the day with the rest of the world. We will hold the programme in the school hall so the rains won’t affect us,” said Anuradha Kamat, principal, Thakur Vidya Mandir.
Jai Hind College, which had planned events for two days, will now celebrate Yoga Day on Sunday. Attendance has not been made compulsory for any students, the college authorities said.
"If rains persist, we might move our celebration to next week," said Manju Nichai, principal, KC College, Churghgate.
Few colleges who had planned events on Saturday have now postponed them to Monday due to heavy rains. "With the BMC advising the colleges to remain shut, we have moved the event to Monday," said Shobhna Vasudevan, principal, Podar College, Matunga.
City tries to keep the date
Celebrations postponed
"Yoga Day activities will be organised next week as our school was shut for two days owing to the rains. There will be separate batches for primary and secondary sections,” said Vira D'Souza, head teacher of primary, Vanita Vishram High School, Charni Road
International schools miss out
International schools such as Cathedral and John Connon School in Fort and Dhirubhai Ambani International School, Bandra, will not be able to celebrate the day as the vacations are on. “Our academic year begins only in mid-July,” said a spokesperson of the Aditya Birla World Academy, Grant Road.
Students asked to do yoga at home
Gundecha Education Academy, Kandivli, has asked students to perform yoga asanas at home on Sunday. “The students have been taught the asanas in school but we do not want to put them through the trouble of coming to school on Sunday. So we have asked them to do the asanas at home and send the videos to us,” said Seema Buch, principal.
Teachers miss training
Most teachers were unable to attend training for Yoga Day organised by the education department on Friday at Dadar because of heavy rains.
White attire compulsory
Many schools have asked students to come dressed in white for the celebrations. Parents of children from a school in Masjid Bandar complained that the school informed them about the dress code at the last minute
Common protocol for Yoga Day
* A circular issued on June 9 from the education department asked schools to hold programmes from 7am to 7.33am on June 21
* Start with a two-minute prayer
* Performing asanas for 15 minutes
* End with pranayam and meditation.
* Schools have to fill in forms stating the number of students, teachers who attended the programme
* The forms also ask them to provide reasons for not holding programmes on that day
(Inputs by Aadesh Bhangre)
First Published: Jun 20, 2015 21:51 IST

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meranu33 comments on Toenails aren't supposed to bleed. But one of mine is.jump to contentmy subredditsannouncementsArtAskRedditaskscienceawwblogbookscreepydataisbeautifulDIYDocumentariesEarthPornexplainlikeimfiveFitnessfoodfunnyFuturologygadgetsgamingGetMotivatedgifshistoryIAmAInternetIsBeautifulJokesLifeProTipslistentothismildlyinterestingmoviesMusicnewsnosleepnottheonionOldSchoolCoolpersonalfinancephilosophyphotoshopbattlespicsscienceShowerthoughtsspacesportstelevisiontifutodayilearnedTwoXChromosomesUpliftingNewsvideosworldnewsWritingPromptsedit subscriptionsfront-all-random | todayilearned-philosophy-Art-WritingPrompts-LifeProTips-television-photoshopbattles-gifs-creepy-movies-UpliftingNews-listentothis-history-explainlikeimfive-TwoXChromosomes-Futurology-books-InternetIsBeautiful-nosleep-Documentaries-gaming-Showerthoughts-DIY-tifu-dataisbeautiful-science-askscience-Fitness-Jokes-worldnews-AskReddit-personalfinance-pics-news-GetMotivated-food-gadgets-mildlyinteresting-funny-IAmA-aww-nottheonion-OldSchoolCool-space-Music-sports-videos-EarthPornmore » WTFcommentsrelatedwant to join? sign in or create an account in seconds|Englishlimit my search to /r/WTFuse the following search parameters to narrow your results:subreddit:subredditfind submissions in "subreddit"author:usernamefind submissions by "username"site:example.comfind submissions from "example.com"url:textsearch for "text" in urlselftext:textsearch for "text" in self post contentsself:yes (or self:no)include (or exclude) self postsnsfw:yes (or nsfw:no)include (or exclude) results marked as NSFWe.g. subreddit:aww site:imgur.com dogsee the search faq for details.advanced search: by author, subreddit...this post was submitted on 27 Jul 20140 points (47% upvoted)shortlink: remember mereset passwordloginSubmit a new linkWTFsubscribeunsubscribe4,507,962 readers8,454 users here now
a community for 7 yearsmessage the moderatorsMODERATORSmastaani625BritishEnglishPoliceKyldeThe JanitorthegrammarunicornWTFGESTAPOYou what?BitterDivorcedDadAnti-KerenskyJack_Shidwtf_modI don't reply to PMs...and 4 more »000Toenails aren't supposed to bleed. But one of mine is. (i.imgur.com)submitted 6 months ago by Elivictus15 commentssharecancelsorry, this has been archived and can no longer be voted onloading...sorted by: bestbesttopnewhotcontroversialoldrandomyou are viewing a single comment's thread.view the rest of the comments →[–]meranu33 0 points1 point2 points 6 months ago (0 children)sorry, this has been archived and can no longer be voted onTea tree oil, 2X daily, applied with a cotton swab for approximately 2 months, will get rid of that!
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From his artfully plated Thai dishes to his handmade carvings that deck the walls, Mr. Kobboon's ample artistic talent flourishes throughout the restaurant. Each morning, he arrives at the restaurant with a bundle of fresh orchids and adorns each table with one of the delicate blooms. Golden lighting keeps the restaurant as warm and inviting as its cuisine's spicy aromas, which waft from the kitchen until 3 a.m. catering to late-night partiers and those trying to stay awake late enough to tuck in the moon. The Gist
“Be patient on the time it takes to get out to the table, it's made fresh to order. It's so worth it!”
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Patent US5552314 - Cloning dna using genetic engineering in host cells - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsMethods and materials for the cloning of DNA, in particular, for the cloning of "unclonable" DNA using genetically engineered host cells. Host cell organisms have been discovered that stabilize and inhibit rearrangement of DNA molecules capable of forming non-standard secondary and tertiary structures....http://www.google.com/patents/US5552314?utm_source=gb-gplus-sharePatent US5552314 - Cloning dna using genetic engineering in host cellsAdvanced Patent SearchPublication numberUS5552314 APublication typeGrantApplication numberUS 08/441,368Publication dateSep 3, 1996Filing dateMay 15, 1995Priority dateJan 8, 1990Fee statusPaidAlso published asCA2033355A1, CA2033355C, DE69033732D1, DE69033732T2, EP0437071A1, EP0681025A1, EP0681025B1, US6017748Publication number08441368, 441368, US 5552314 A, US 5552314A, US-A-5552314, US5552314 A, US5552314AInventorsAlan L. GreenerOriginal AssigneeStratageneExport CitationBiBTeX, EndNote, RefManPatent Citations (2), Non-Patent Citations (20), Referenced by (12), Classifications (17), Legal Events (13) External Links: USPTO, USPTO Assignment, EspacenetCloning dna using genetic engineering in host cellsUS 5552314 AAbstract Methods and materials for the cloning of DNA, in particular, for the cloning of "unclonable" DNA using genetically engineered host cells. Host cell organisms have been discovered that stabilize and inhibit rearrangement of DNA molecules capable of forming non-standard secondary and tertiary structures. Organisms are engineered to contain at least one mutation which inactivates homologous recombination and at least one mutation in a DNA repair pathway. Examples of such DNA pathways include UV repair pathway, the SOS repair pathway, the mismatch repair pathway, the adaptive response pathway, the heat shock response pathway, the osmotic shock response pathway, the repair pathway of alkylation damage, the repair pathway of uracil incorporation into DNA and pathways involved in maintaining DNA superhelicity. The host organisms of this invention are suitable for cloning DNA capable of forming non-standard and tertiary structures such as found in eukaryotic DNA.
I claim: 1. A culture of E. coli having ATCC deposit number 55695 wherein the E. coli is recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, (MsdRMS), endA1, gyrA96, thi, relAl, lac, supE44, and {F'pro AB, lacIQ ZM15, Tn1056 and the E. coli can prevent rearrangements in DNA sequences containing inverted repeats or Z-DNA.
This is a continuation of application Ser. No. 08/051,572, filed Apr. 22, 1993, now abandoned, which is a continuation of application Ser. No. 07/462,505 filed on Jan. 8, 1990, now abandoned.
This invention relates to certain methods and materials for the cloning of DNA and, in particular, to the cloning of "unclonable" DNA using a genetically engineered host cell. This cloning host organism includes particular mutations from existing cells which have been found to stabilize tester DNA plasmids, and can be assembled using standard transducing and plating techniques.
The ability to elucidate gene structure and function often depends to a great extent upon the construction of recombinant DNA libraries accurately representing the total genome of a subject organism, followed by the cloning of this recombinant DNA. Generally, the total DNA of cells from the subject organism is isolated and cut into fragments using specific restriction enzymes. The fragmented DNA sequences are then inserted into appropriate vectors for subsequent propagation and amplification in a foreign host. Commonly used vectors include plasmids, cosmids or phages. Examples of host cells suitable for propagation of foreign DNA sequences include bacteria (i.e., E. coli, Bacillus subtilin, Pseudomonas aeruginosa, yeast (i.e., Saccharomyces cerevisiae) Drosophilia (i.e., Drosophilia melongaster) mammalian cell lines (CHO, L-cells) human cell lines (i.e., Hela, Baculovirus, plant cell lines.
To date a number of obstacles have limited the establishment or stable inheritance of foreign (non-native) DNA in host cells. One barrier to establishment of foreign DNA is the presence of restriction endonuclease that cleave such DNA. See, e.g., Bickle, T. (1982) In: Nucleases (Linn, S. M., and Roberts, R. J., eds.). Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; Raleigh, E. A., and Wilson, G. (1986) Proc. Natl. Acad. Sci. USA 83:9070; and Heitman, J., and Model, P. (1987) J. Bact. 169:3243. Genetic inactivation of these restriction enzymes in certain host cells (restriction minus strains) can result in a more efficient introduction of foreign DNA via transformation, transduction, electroporation or conjugation.
Despite such genetic manipulation of host cells, however, many DNA fragments, particularly those from eucaryotic organisms, have been deemed "unclonable." Many of these "unclonable" DNA segments are known to contain sequences capable of forming non-standard secondary and tertiary structures. See, e.g., Erlich, D. (1989) In: Mobile DNA (Berg, D. E. and Howe, M. M., eds.). ASM Publications, Washington D.C. and Santella, R. M., Grunberger, D., Weinstein, I. B., and Rich, A. (1981) Proc. Natl. Acad. Sci. USA 78:1451. DNA that is capable of forming cruciforms (hydrogen bonded hairpin structures formed from inverted repeat sequences) and Z-DNA (a left handed zig zag configured DNA resulting from alternating purine-pyrimidine residues), for example, is rapidly deleted or rearranged in E. coli. See, e.g., Santella, R. M., Grunberger, D., Weinstein, I. B., and Rich, A. (1981) Proc. Natl. Acad. Sci. USA 78:1451 and Fuchs, R. P. P., Freund, A. M., and Bichara, M. (1988) In: Methods and Consequences of DNA Damage Processing (Friedberg, E. C. and Hanawalt, P. C., eds.). Alan R. Liss, Inc., N.Y.
Previous attempts to increase the stability of cloning such complex DNA structures has met with only limited success. See Wyman, A., Wertman, K., Barker, D., Helms, C., and Petri, W. (1986) Gene 49:263-271. Wyman et al. reported that certain mutations in E. coli host cells resulted in an increased fidelity of representation of certain complex eucaryotic DNA sequences. Specifically, this group found that the use of E. coli strains with mutations in homologous recombination pathways (recB, recC, and sbcB, or recD) increased the representation of polymorphic sequences of genomic DNA. While these authors did not fully characterize the polymorphic nature of the DNA sequences cloned, the subject sequences were thought to contain long segments of inverted repetitions or palindromes.
Chalker et al. have reported that the mutation of a single E. coli gene involved in a secondary pathway of homologous recombinations (sbcC) results in the stable propagation of a long palindromic DNA sequence. Chalker, A., Leach, D., and Lloyd, R. (1988) Gene 71:201-205. In marked contrast to the study of Wyman et al., however, the Chalker group did not observe any major effects of mutations in the primary pathway of homologous recombination on palindrome stability. Ishiura et al. studied the effects of mutant strains of E. Coli on the deletion of genomic eucaryotic DNA during cloning. Ishiura, M., Hazumi, N., Koide, T., Uchida, T., and Okada, Y. (1989) J. Bact. 171:1068-1074. These authors report that only a quadruple combination of host mutations (recB, recC, sbcB and either recJ or recN) prevented the deletion of DNA segments during host propagation. Strains with single mutations in either primary or secondary pathways of homologous recombination or combined mutations in recombination and restriction pathways did not prevent deletion. There were no data on the structural configuration of the DNA sequences employed.
The data from these studies, taken together, indicate that host cell mutations of recombination pathways have not lead to increased stability of certain forms of complex DNA during propagation, and the divergent and often conflicting results noted above indicate that, at present, there is not an identifiable or even ascertainable host strain that is suitable for cloning complex, eucaryotic DNA.
The present inventor has discovered that complex DNA sequences can be stabilized during cloning by utilizing hosts with combined mutations in certain nucleic acid recombination and repair pathways. Described herein are novel bacterial hosts designed for cloning foreign DNA by combining DNA repair mutations into host strains deficient in homologous recombination. Mutations which allow for stabilization of tester plasmids containing complex DNA are identified in existing strains and then assembled using standard techniques of transduction, screening, selection and propagation. The resulting bacteria can be used to stabilize and clone complex DNA such as contained in eucaryotic genomes.
SUMMARY OF THE INVENTION This invention provides for the genetic construction of host organisms that provide a stable environment for cloning foreign DNA molecules capable of assuming secondary and tertiary structure susceptible of rearrangement. These hosts are, thus, suitable for cloning DNA molecules capable of assuming such secondary and tertiary structures. The host organisms of this invention provide a stable environment for DNA molecules containing inverted repeat sequences, capable of forming cruciforms, and DNA molecules containing alternating purine-pyrimidine residues, capable of forming Z-DNA. The host organisms of the invention are characterized as being recombination deficient and containing genetic mutations in DNA repair pathways. In a preferred embodiment, this invention provides for an E. coli host of the genotype recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, hsdRMS, endA1, gyrA96, thi, relA1, lac, supE44, {F'proAB, lacIQ ZM15, Tn10}.
The invention further provides for a method of cloning structurally complex DNA, such as found in the eucaryotic genome. This method comprises isolating DNA from an organism, cutting the DNA into fragments, inserting DNA fragments of that organism into a vector, and introducing the vector in the insert into the host organism of this invention. The method involves the use of a host characterized as recombination deficient with mutations in DNA repair systems. In a preferred embodiment, the E. coli host used in cloning is of the genotype recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, hsdRMS, endA1, gyrA96, thi, relA1, lac, supE44, {F'proAB, lacIQ ZM15, Tn10}.
This invention further provides for a method of constructing a host organism suitable for cloning DNA molecules capable of assuming secondary and tertiary structures susceptible of rearrangement. The method comprises identifying mutations of recombination and DNA repair pathways which stabilize DNA in existing hosts and assembling these mutations using standard genetic techniques. This invention further provides for construction of a novel strain of E. coli, designated SURE�. Construction of the SURE� strain is accomplished by successively transducing E. coli ER1451 with bacterial genomes containing mutations in recombination and DNA repair systems. A preferred method of constructing the SURE� strain is outlined in FIG. 2 and further described herein.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 2 is a flow diagram of the steps used to construct a strain of E. coli that is suitable for cloning DNA capable of forming secondary and tertiary structures susceptible of rearrangement. The SURE� strain was assembled from ER1451 using sequential steps of P1 transduction (P1) Bochner plating, and conjugal mating. The E. coli strain designation for each P1 transduction step indicates the source of genetic material transduced. The genotypes of these strains are set forth in Table 1.
FIG. 1 is a schematic representation of the recombination pathways of E. coli.
FIG. 3 is a schematic gene map of the inverted repeat tester plasmid, designated pAL28α.
FIG. 4 is a schematic gene map of the Z-DNA tester plasmid system, designated pAL20Z.
DETAILED DESCRIPTION OF THE INVENTION I. Abbreviations--the following abbreviations are used throughout the specification and claims.
A. Relevant Genotypes
end--endonuclease
hsd--host specificity restriction system
mcr--restriction system for DNA containing methylated cytosine residues
mrr--restriction system for DNA containing methylated adenine residues
recBC--Exonuclease V. involved in primary homologous recombination pathway
sbcC--suppressor of recBC
thr--threonine
umuC--uv mutagenesis
uvr--ultraviolet repair (excision repair system)
recJ--exonuclease involved in secondary homologus recombination pathways
lac--an uncharacterized mutation within the lac operon which results in colonies more intensely blue in the presence of the lacZΔM15 mutation and a source of the α-complementation lac segment
E. coli--Escherichia coli bacteria
SOS--genes coding for components of DNA repair systems; inducible by DNA damage
II. Definitions--Unless otherwise indicated, the terms listed below will be defined as set forth.
cruciform--hydrogen bonded hairpin structures formed from inverted repeat sequences of DNA
infection--any process used to introduce DNA into host organisms. Includes transduction, transformation transfection, conjugal mating, and electroporation.
insertion--any process used to integrate foreign DNA into vectors inverted repeats--completely or partially identical but oppositely oriented DNA sequences restriction minus--cell strains lacking genes coding for enzymes which break down foreign DNA
vector--autonomously replicating DNA units into which DNA fragments are inserted for cloning
Z-DNA--a left-handed helical configuration of DNA resulting from alternating purine-pyrimidine residues
Construction of mutant bacterial strain DNA molecules, capable of assuming nonstandard secondary and tertiary structures, as commonly found in eucaryotic genomes, are often completely deleted or rearranged during propagation in cloning host cells. A major reason for this lack of fidelity in cloning may be the existence in the host cell of mechanisms related to the replication and repair of DNA. Examples of such mechanisms include: (1) pathways designed for homologous or non-homologous recombination; (2) restriction systems which cleave foreign DNA sequences; (3) endonucleases which catalyze the breakdown of DNA; and (4) systems designed for repair of DNA. Both procaryotic and eucaryotic organisms have developed an extensive array of systems designed for DNA repair. Examples of such repair systems include: uv repair pathway; SOS repair pathway; mismatch repair; adaptive response; heat shock response; osmotic shock response; repair of alkylation damage; repair of uracil incorporation into DNA; and gene products involved in maintaining DNA superhelicity. The role of such repair systems in cloning of foreign DNA has not yet been investigated. As noted above, prior attempts to increase stabilization of foreign DNA have focused only on host cell mutations in the recombination and/or restriction pathways.
The inventors have engineered a host organism with mutations in repair as well as recombination pathways. The resulting host cells serve to stabilize various complex forms of DNA during propagation.
The new strain has been designed to be a superior host for optimizing the construction of cDNA and genomic DNA libraries as well as for increasing the probability that the individual clones obtained will accurately reflect the genetic makeup of the subject organism. The construction of host strains with combined mutations was accomplished by crossing or mating strains with individual mutations in metabolic pathways. Means of constructing new strains by DNA introduction or mutation include, but are not limited to, Hfr mating, generalized transduction, specialized transduction, conjugation, transposon mutagenesis, oligonucleotide-directed mutagenesis, and transformation followed by genetic recombination. Examples of transducing bacteriophages include, but are not limited to, P1 or lambda. Details of a preferred method of construction using P1 phage transduction are presented in Example 1. Screening and identification of mutant strains incorporating and expressing the transduced foreign DNA can be accomplished by any number of methods. Examples of such identification and screening methods include, but are not limited to, screening for phenotypic traits such as antibiotic sensitivity, drug resistance, or dependency on nutrient sources.
The parent strain, used as the basis for construction, can be any existing strain with a well-defined genotype that preferably includes at least one of the specific mutations sought to be present in the final genotype. Numerous such strains are available and have been well characterized. See, e.g., Ishiura, M., Hazumi, N., Koide, T., Uchida, T., and Okada, Y. (1989) J. Bact. 171:1068-1074. The parent strain is then transduced with or mated with DNA from a second strain known to contain at least one additional desired mutation. Those progeny cells containing the desired mutations from both the donor and recipient cell are identified, propagated and used through subsequent transduction or mating steps. Once the organism is identified which contains all of the desired mutations, such an organism can be additionally manipulated to make it suitable with a variety of vectors and screening procedures. The invention is a propagatable cell containing combined mutations in both recombination and DNA repair systems. Preferably, the cloning host cell also contains mutations in the endonuclease and restriction systems, which mutations increase the utility of host cells for cloning purposes.
In a preferred embodiment, the inventors have engineered and constructed a novel strain of E. coli, with combined mutations in recombination and repair systems, that stabilizes complex foreign DNA during propagation. E. coli strain ER1451, known to have mutations of certain DNA endonucleases and components of the restriction pathway, was sequentially transduced with bacterial genomes having the mutations sought to be introduced. In each case, the transduced DNA also contained a gene for tetracycline resistance. Following each individual transduction step, strains containing the combined mutations were screened on Bochner plates in order to isolate tetracycline sensitive derivatives which would then serve as a recipient for the next transduction step. Successive transduction steps can be performed in any order. In a preferred embodiment, the ER1451 strain was transduced sequentially with DNA containing mutations in the recombination pathway, ultraviolet repair pathway, SOS repair pathway, the restriction pathway, a tetracycline-resistant episome carrying a blue/white screening cassette and a lactose sensitivity gene. As a result of these sequential transduction and screening procedures, a strain of bacteria was created that is recombination deficient, restriction minus, endonuclease deficient, and with mutations in DNA repair systems. Details of this preferred construction sequence are given in Example 1. The resulting bacterial host, constructed by this method, is known as SURE, a trademark of Stratagene Cloning Systems.
The SURE� strain constructed by this method was then tested to determine its effect on the stability of cloned DNA sequences capable of forming secondary and tertiary structures and susceptible to rearrangement during propagation. Examples of such DNA sequences include, but are not limited to inverted repeats and alternating purine-pyrimidine residues.
Initial studies were performed to evaluate the effects of the SURE� strain on the stability of inverted repeat DNA sequences, capable of forming structures known as cruciforms. An inverted repeat plasmid tester system was created by modifying plasmid pBR322. This tester plasmid system, designated pAL28α, used chloramphenicol resistance as the inverted repeat sequence. A variety of strains of E. coli, existing strains as well as the SURE� strain, were infected with a tester inverted repeat plasmid. Inverted repeats were found to be rapidly rearranged in virtually all existing strains tested. In contrast to the results with existing strains, however, the SURE� strain was found to provide a stabilizing environment for these inverted repeat sequences. The percentage of rearrangement was decreased 20 fold in the SURE� strain when compared to the other strains tested. The SURE� strain was, therefore, engineered to carry mutations blocking pathways responsible for repairing DNA lesions. Preferred specific mutations for blocking these two key pathways are uvrC and umuC respectively, although mutations in other genes of this pathway or mutations in other repair pathways would give the desired effect. This presence of these mutations was demonstrated to result in a 10 to 20 fold increase in stability of DNA containing long inverted repeats.
Z-DNA tester plasmids were created as derivatives of plasmid pBR322. The pBR322 plasmid, containing sequences for tetracycline and penicillin resistance, was modified to include a Z-DNA segment in the promoter region for chloramphenicol resistance. The newly created tester plasmid system was designated pAL20Z. This tester DNA plasmid was infected into existing strains of E. coli as well as the newly constructed SURE� strain. The Z-DNA containing segments were quickly deleted in the existing strains while, in the SURE� strain, deletion was significantly and markedly reduced. These data demonstrate that a combination of mutations in recombination and repair pathways, such as engineered in the SURE� strain, provide a stabilizing influence to DNA capable of forming nonstandard secondary and tertiary structures.
In E. coli, homologous recombination is a complex, multicomponent process that can proceed by three interdependent pathways (see FIG. 1). These 3 pathways, recBCD, recE and recF, all require the recA gene product and a set of accessory proteins. A standard recA host, therefore, is completely deficient in homologous recombination. Recombination in a recB strain (the recBCD pathway is the primary pathway in a wild type E. coli) is reduced to approximately 0.5%. The residual activity is due to the presence of the recE and recF alternate pathways which do not involve recB. A recB recJ double mutant, however, is virtually identical to a recA strain in its recombination deficiency; recJ is required for both alternate pathways. To model this, recombination can be blocked in host organisms with a variety of mutations. In addition to the ones set forth above, examples of such alternate mutations include recB, recO and recB, recJ, recN.
The inventors discovered that insertion of either the recA or recB mutations into a sbcC, recJ, umuC, uvrC strain, stabilized both Z-DNA and inverted repeats. The recB derivative was found to exhibit superior stabilization effects. This discovery, that combined mutations in recombination and DNA repair pathways, stabilize complex DNA during propagation is significant.
The cloning host organism of the instant invention is restriction minus, preferably carrying, in E. coli, hsd, mcr and mrr mutations. The absence of mcr and mrr restriction activity increases the size and representation of libraries constructed with methylated or hemimethylated DNA. The absence of hsd activity increases the size and representation of libraries constructed from sequences containing EcoK recognition sequences. An end mutation (inactivating a DNA endonuclease) was observed to result in improved quality of plasmid DNA minipreps.
In a preferred embodiment, the host cell of this invention also harbors a tetracycline-resistant F'episome carrying the laclQ ZM15 cassette making it suitable for blue/white screening on plates supplemented with X-gal and IPTG. The uncharacterized mutation present in XL1-Blue cells, which makes both plaques and colonies more intensely blue when a source of the α-complementation fragment is introduced, has been added. The cell is, thus, suitable for plasmid or phage libraries using a variety of vectors.
The following examples are set forth to assist in understanding the invention and should not, of course, be construed as specifically limiting the invention described and claimed herein. Such variation of the invention which would be within the purview of those in the art, including the substitution of all equivalents now known or later developed, are to be considered to fall within the scope of the invention as hereinafter claimed.
EXAMPLE 1 Construction of an E. coli strain.
A novel strain of E. coli was constructed from existing strains utilizing standard transduction and plating techniques. In this example, transduction with P1 phage particles was utilized. Bochner plating (Bochner, et al., J. Bacteriol., 143:926, 1980) was then used to screen derivatives for the desired genotype. An outline of the construction method is schematically diagramed in FIG. 2. The genotypes of the various E. coli strains used in the construction pathway are set forth in Table 1.
TABLE #1______________________________________Strain  Genotypes______________________________________ER1451  lac-proAB, thi, gyrA96, endA1, hsdR17, relA1,   supE44, {F' traD36, proAB, lacIQ Z M15}CES229  recD1009, hsdR, sbcC201, phoR79::Tn10, recAJC12166 recB21, recC22, sbcB15, sbcC201, thr-1, leuB6, thi-1,   lacY1, galk2, ara-14, xyl-5, mtl-1, proA2, his-4,   argE3, rpsL31, tsx-33, supE44, recJ284::Tn10CAG12156   MG1655 (E. coli K-12 F-  λ) uvrC279::Tn10JC8947  AB1157 (standard E. coli K-12) umuC::Tn5AG279   same as JC12166 except recJ+, recF143, fuc::Tn10JH122   GW1000 (standard E. coli K12, mcrB4::Tn10,   mrr2::Tn5, hsdR2)BB4     hsdR514, supE44, supP58, lacY1, galK2, galT22,   metB1, trpR55, {F'(lacIQ Z M15::Tn10}AG239   thi, gyrA, supE44, lac- (uncharacterized) TetRAG1     recA, endA1, gyrA96, thi, hsdR17, supE44, relAC600    supE44, thi-1, leuB6, lacY1, tonA21, mcrANM621   recD, hsdR, mcrA, mcrB, supEHB101   hsdS20, supE44, ara14, galK2, lacY1, proA2,   rpsL20, xyl-5, recA13, mcrBTBI     ara, lac-proAB, rpsL, 80, lacZ M15, hsdRDH5α   recA1, endA1, relA1, thi, hsdR17, supE44, gyrA96______________________________________
ER1451 cells were grown at 37� C. to a density of 5�108 to 1�109 cells/ml in media containing tryptone, yeast extract, NaCl and agar (LB media) containing 5mM CaCl2. Cells were harvested by centrifugation and resuspended in 100 mM MgSO4, 5 mM CaCl2 (MC media) media at 37� C. for 15 minutes. Cells were again harvested by centrifugation and resuspended in 1/10 the original volume of MC media. 0.1 ml of the resulting cell suspension was mixed with a P1 phage, containing the genome of E. coli strain CES229, and incubated at 37� C. for 15 minutes. The reaction was terminated by the addition of 0.1 ml of 0.2M sodium citrate. 0.5 ml of LB media was added and cultures grown at 37� C. for an additional 60 minutes. These incubation conditions were found to be sufficient for expression of transduced DNA. Cells (5�106) were then plated directly onto LB plates containing 15 μg/ml tetracycline. Transductants were screened for the desired phenotype, then plated onto Bochner plates and screened for tetracycline sensitivity. The culture media for this screening procedure contained in final concentration: 10 g/L tryptone, 5 g/L yeast extract, 15 g/L agar, 10 g/L NaCl, 10 g/L NaH2 PO4, 2 g/L glucose, 12 mg/L fusaric acid, 0.1 mM ZnCl2, and 50 mg/L of chlortetracycline. Cell colonies demonstrating a rapid rate of growth and sensitivity to tetracycline were identified, recovered and utilized for subsequent transduction as indicated in FIG. 2. ER1451 cells were sequentially transduced with P1 phages containing the genome of E. coli strains CES229, JC12166, CAG12156, JC8947, AG239, AG279, and JH122. As a result of these transductions, the following genetic mutations were assembled into the original ER1451 strain: phoR; sbcC201; recJ; uvrC; umuC; lac*; fuc; recB21; mcrB and mrr. The resulting E. coli strain, AG324, was subjected to F' mating for the purpose of introducing a tetracycline resistant F'episome carrying a lacIQ ZM15 cassette for use in blue/white screening. The final E. coli strain (ATCC deposit number 55695) constructed has been named SURE�, a trademark of Stratagene Cloning Systems. The genotype of SURE� is recB, recJ, sbcC201, phoR, uvrC, umuC::Tn5, mcrA, mcrB, mrr, Δ(hsdRMS), endA1, gyrA96, thi, relA1, lac*, supE44, {F'proAB, lacIQ ZM15, Tn10}. The SURE� strain of E. coli was then used to test for stability of various structurally complex DNA sequences.
EXAMPLE 2 Stability of Inverted Repeats
DNA molecules containing an inverted repeat sequence coding for chloramphenicol resistance were created and inserted into modified pBR322 plasmid vectors. A gene map of the inverted repeat tester system, designated plasmid pAL28α, is schematically diagramed in FIG. 3. Numerous strains of E. coli, including the SURE� strain, were transformed with pAL28α. Transformants were inoculated in media containing only penicillin and grown for up to 100 generations (with serial dilutions). After every 10 or 20 generations, aliquots of cells were screened on either penicillin containing or chloramphenicol containing plates. Cells in which the inverted repeat chloramphenicol resistant sequences were rearranged lost their chloramphenicol resistance. Percentage of cells generating rearrangement of the tester plasmid could, therefore, be readily determined by monitoring chloramphenicol sensitivity. Results from eight of the strains studied are summarized in Table 2. The genotypes of the strains reported in Table 2 are set forth in Table 1 above.
TABLE #2______________________________________STABILITY OF INVERTED REPEATSIN VARIOUS E. COLI STRAINS       Percentage of cells       generating rearrangedStrain      plasmid per generationa______________________________________AG1         15-20DH5α  15-20C600        20-25NM621       20-25HB101       15-20TB1         20-25DL538       20-25SURE �   0.8-1.2______________________________________ a Percent rearrangement calculated from the number of cells maintaining the inverted repeat tester plasmid in its unrearranged configuration (chloramphenicol resistant) divided by the total number of plasmidcontaining cells.
It is evident from the data in Table 1 that only the SURE� E. coli strain markedly reduced DNA rearrangement. The internal environment of the SURE� strain, therefore, provides a stabilizing influence on inverted repeat DNA sequences. Note that each of the E. coli strains tested contained at least some of the mutations found in the SURE� strain. It is important to note here that even though some strains (i.e,, DH5α) contained mutations in different metabolic pathways (homologous recombination, restriction pathway and endonuclease), such combined mutations were not effective in diminishing the rate of rearrangement. Because the presence of these individual mutations in other strains did not prevent rearrangement of the inverted repeat DNA sequence, it appears as though it is the combination of repair and recombination mutations in SURE� that is responsible for increased stability.
EXAMPLE 3 Stability of Z-DNA
DNA molecules were created which contained alternating purine and pyrimidine residues capable of forming Z-DNA. These Z-DNA containing sequences were inserted into a modified pBR322 plasmid designated pAL20Z (see FIG. 4). A sequence of 26 alternating purine-pyrimidine residues (GC) were inserted into and blocked expression of the promoter region for chloramphenicol resistance. Because of the absence of a promoter in the intact plasmid, host cells containing pAL20Z demonstrate chloramphenicol sensitivity. Deletion of the repeating sequences results in reconstitution of the promoter and, therefore, expression of chloramphenicol resistance. A variety of E. coli host strains were transformed with pAL20Z. Transformants were inoculated into media containing penicillin and grown for up to 200 generations (with serial dilutions). After every 20 generations, 106 cells were plated onto media containing chloramphenicol (50 μg/ml). The percentage of cells demonstrating chloramphenicol resistance was taken as a direct reflection of the percent of cells rearranging the tester plasmid. The results from transformation of seven of these E. coli strains, including the SURE� strain, are shown below in Table 3. The genotypes of these E. coli strains can be found in Table 1.
TABLE #3______________________________________STABILITY OF Z-DNA IN VARIOUSE. COLI STRAINS         Percentage of cells         generating rearrangedStrain        plasmids per 25 generationsa______________________________________AG1           15DH5α    15C600          10NM621         20HB101         15TB1           10SURE � strain         <0.1______________________________________ a Percent ZDNA rearrangement calculated by determining the percentag of chloramphenicol resistant cells (indicative of a specific ZDNA rearrangement) within a population after 25 generations.
It is evident from these data that the SURE� E. coli strain significantly and markedly reduced the amount of DNA rearrangement. The SURE� strain, therefore, provides a stabilizing environment for Z-DNA. Taken together, the data from Examples 2 and 3 demonstrate that a cloning host organism can be engineered and constructed such that it provides a stabilizing environment for complex DNA molecules capable of forming secondary and tertiary structures. Furthermore, the data demonstrate that this host organism is effective in inhibiting rearrangements of DNA molecules capable of forming a variety of such secondary and tertiary structures.
A novel strain of bacteria has been engineered by combining a number of mutations which significantly reduce the rate of both homologous and non-homologous recombination within segments of cloned foreign DNA. Non-bacterial DNA, particularly from eucaryotic sources, frequently contain sequences capable of forming secondary and tertiary structures such as cruciforms (due to presence of inverted repeats) or Z-DNA (found in alternating purine-pyrimidine stretches). These structures are highly recombinogenic in standard bacterial cloning hosts which are incapable of carrying out homologous recombination. Mutations which eliminate certain repair pathways responsible for excision of or replication past DNA lesions greatly stabilize these unusual structures. By engineering mutations which eliminate these pathways into a host strain deficient in homologous recombination, a novel host that is suitable for cloning foreign DNA has been constructed.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4190495 *Sep 27, 1976Feb 26, 1980Research CorporationModified microorganisms and method of preparing and using sameUS4748119 *Jan 15, 1987May 31, 1988Alexander RichProcess for altering and regulating gene expression* Cited by examinerNon-Patent CitationsReference1 *Bachmann B. 1990, Microbiol. Rev. 54, 130 197.2Bachmann B. 1990, Microbiol. Rev. 54, 130-197.3 *Bachmann, B. 1983. Microbiol. Rev. 47, 180 230.4Bachmann, B. 1983. Microbiol. Rev. 47, 180-230.5 *Bullock et al. 1987 Bio Techniques 5, 376 378.6Bullock et al. 1987 Bio Techniques 5, 376-378.7 *Chalker et al. 1988 Gene 71, 201 205.8Chalker et al. 1988 Gene 71, 201-205.9 *Friefelder, D. 1983, in: Molecular Biology. A Comprehensive Introduction to Prokaryotes and Eukaryotes. Science Books Int l, (Boston)/Van Nostrand Reinhold Co. (New York), pp. 145 148, 730 837.10Friefelder, D. 1983, in: Molecular Biology. A Comprehensive Introduction to Prokaryotes and Eukaryotes. Science Books Int'l, (Boston)/Van Nostrand Reinhold Co. (New York), pp. 145-148, 730-837.11 *Gerhardt et al. (eds.) 1981. in: Manual of Methods for General Bacteriology Am. Soc. for Microbiol., Washington D.C. pp. 243 265.12Gerhardt et al. (eds.) 1981. in: Manual of Methods for General Bacteriology Am. Soc. for Microbiol., Washington D.C. pp. 243-265.13 *Heitman et al. 1987. J. Bacteriol. 169, 3243 3250.14Heitman et al. 1987. J. Bacteriol. 169, 3243-3250.15 *Ishiura et al. 1989 Proc. Nat l Acad. Sci. USA. 171, 1068 1074.16Ishiura et al. 1989 Proc. Nat'l Acad. Sci. USA. 171, 1068-1074.17 *Kolodner et al. 1985. J. Bacterial. 163, 1060 1066.18Kolodner et al. 1985. J. Bacterial. 163, 1060-1066.19 *Raleigh et al. 1986 Proc. Nat l. Acad. Sci. USA 83, 9070 9074.20Raleigh et al. 1986 Proc. Nat'l. Acad. Sci. USA 83, 9070-9074.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS6017748 *Jun 7, 1995Jan 25, 2000StratageneCloning host organismsUS7198924Mar 4, 2004Apr 3, 2007Invitrogen CorporationMethods and compositions for synthesis of nucleic acid molecules using multiple recognition sitesUS7223576Mar 10, 2004May 29, 2007Invitrogen CorporationExpression vector which utilizes site specific recombinases as insertion tools during in vivo and/or in vitro gene transferUS7282326Jan 30, 2002Oct 16, 2007Invitrogen CorporationRecombinational cloning using engineered recombination sitesUS7304130Nov 2, 1999Dec 4, 2007Invitrogen CorporationVector for use as a propagation tool in genetic engineeringUS7393632Dec 11, 2000Jul 1, 2008Invitrogen Corp.Use of multiple recombination sites with unique specificity in recombinational cloningUS7408049Jul 19, 2004Aug 5, 2008Invitrogen CorporationExpression vector comprising topisomerase site-specific recombination sequences for use as gene transfer and propagation tools in bioreactors; gene therapy; protein and gene expression analysisUS7670823Mar 2, 2000Mar 2, 2010Life Technologies Corp.Compositions for use in recombinational cloning of nucleic acidsUS7714116Sep 19, 2008May 11, 2010Life Technologies CorporationExpression vector comprising topisomerase site-specific recombination sequences for use as gene transfer and propagation tools in bioreactors; gene therapy; protein and gene expression analysisUS8241896Dec 29, 2009Aug 14, 2012Life Technologies CorporationCompositions for use in recombinational cloning of nucelic acidsUS8685893Jul 28, 2008Apr 1, 2014Genentech, Inc.Phage displayWO2000052141A1 *Mar 2, 2000Sep 8, 2000Life Technologies IncCells resistant to toxic genes and uses thereof* Cited by examinerClassifications U.S. Classification435/252.33, 435/320.1, 435/252.3International ClassificationC12N15/70, C12N15/09, C12R1/19, C12N15/63, C12N1/21, C07K14/245Cooperative ClassificationC07K14/245, C12N15/63, C12R1/19, C12N15/70European ClassificationC12N15/70, C07K14/245, C12R1/19, C12N15/63Legal EventsDateCodeEventDescriptionJul 6, 2010ASAssignmentOwner name: AGILENT TECHNOLOGIES, INC.,COLORADOFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:STRATAGENE CALIFORNIA;REEL/FRAME:24630/870Effective date: 20100615Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:STRATAGENE CALIFORNIA;REEL/FRAME:024630/0870Owner name: AGILENT TECHNOLOGIES, INC., COLORADODec 10, 2008ASAssignmentOwner name: STRATEGENE CALIFORNIA, CALIFORNIAFree format text: RELEASE BY SECURED PARTY;ASSIGNOR:MERRILL LYNCH BUSINESS FINANCIAL SERVICES, INC.;REEL/FRAME:021952/0174Effective date: 20081030Feb 15, 2008FPAYFee paymentYear of fee payment: 12Mar 3, 2004FPAYFee paymentYear of fee payment: 8Feb 9, 2004ASAssignmentOwner name: TCW SPECIAL CREDITS FUND V - THE, CALIFORNIAFree format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA, FORMERLY KNOWN AS STRATAGENE;REEL/FRAME:014981/0660Effective date: 20040203Owner name: TCW SPECIAL CREDITS FUND V - THE PRINCIPAL FUND -Free format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA, FORMERLY KNOWN AS STRATAGENE /AR;REEL/FRAME:014981/0660Feb 6, 2004ASAssignmentOwner name: TCW SPECIAL CREDITS FUND V-THE PRINCIPAL FUND C/OFree format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA, FORMERLY KNOWN AS STRATAGENE;REEL/FRAME:014981/0654Effective date: 20040203Free format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA, FORMERLY KNOWN AS STRATAGENE /AR;REEL/FRAME:014981/0654Feb 4, 2004ASAssignmentOwner name: MERILL LYNCH BUSINESS FINANCIAL SERVICES INC., ILLFree format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA;REEL/FRAME:015167/0632Effective date: 20040121Owner name: MERILL LYNCH BUSINESS FINANCIAL SERVICES INC. 222Free format text: SECURITY AGREEMENT;ASSIGNOR:STRATAGENE CALIFORNIA /AR;REEL/FRAME:015167/0632Owner name: MERRILL LYNCH BUSINESS FINANCIAL SERVICES INC. 222Free format text: A CORRECTIVE SECURITY AGREEMENT TO CORRECT THE ASSIGNOR ON REEL 014926 FRAME 0078;ASSIGNOR:STRATAGENE CALIFORNIA /AR;REEL/FRAME:014943/0500Jan 29, 2004ASAssignmentOwner name: STRATAGENE, CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:UNION BANK OF CALIFORNIA, N.A.;REEL/FRAME:014885/0317Effective date: 20040115Free format text: RELEASE OF PATENTS COLLATERAL;ASSIGNOR:UNION BANK OF CALIFORNIA, N.A.;REEL/FRAME:014918/0364Effective date: 20040123Owner name: STRATAGENE 11011 N. 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$11.99 KJV Old Scofield Study Bible, Large Print, Bonded leather, Black, Thumb-Indexed
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Talk:2009–10 Louisville Cardinals men's basketball team - Wikipedia
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Mike Tyson's deserted mansion, complete with wall to wall zebra carpet and an ice cream sundae bar! : picsjump to contentmy subredditsannouncementsArtAskRedditaskscienceawwblogbookscreepydataisbeautifulDIYDocumentariesEarthPornexplainlikeimfiveFitnessfoodfunnyFuturologygadgetsgamingGetMotivatedgifshistoryIAmAInternetIsBeautifulJokesLifeProTipslistentothismildlyinterestingmoviesMusicnewsnosleepnottheonionOldSchoolCoolpersonalfinancephilosophyphotoshopbattlespicsscienceShowerthoughtsspacesportstelevisiontifutodayilearnedTwoXChromosomesUpliftingNewsvideosworldnewsWritingPromptsedit subscriptionsfront-all-random | sports-GetMotivated-creepy-explainlikeimfive-DIY-AskReddit-news-personalfinance-food-Jokes-Music-worldnews-aww-Fitness-Documentaries-videos-history-LifeProTips-listentothis-mildlyinteresting-television-TwoXChromosomes-nosleep-movies-books-Showerthoughts-askscience-WritingPrompts-space-EarthPorn-todayilearned-InternetIsBeautiful-UpliftingNews-Art-funny-photoshopbattles-science-gaming-philosophy-dataisbeautiful-gifs-IAmA-pics-nottheonion-Futurology-OldSchoolCool-gadgets-tifumore » picscommentsrelatedother discussions (4)want to join? login or register in seconds|Englishlimit my search to /r/picsuse the following search parameters to narrow your results:subreddit:subredditfind submissions in "subreddit"author:usernamefind submissions by "username"site:example.comfind submissions from "example.com"url:textsearch for "text" in urlselftext:textsearch for "text" in self post contentsself:yes (or self:no)include (or exclude) self postsnsfw:yes (or nsfw:no)include (or exclude) results marked as NSFWe.g. subreddit:aww site:imgur.com dogsee the search faq for details.advanced search: by author, subreddit...this post was submitted on 17 Dec 20123 points (63% upvoted)shortlink: remember mereset passwordloginSubmit a new linkpicsunsubscribesubscribe6,778,869 readers8,219 users here nowA place to share photographs and pictures. Feel free to post your own, but please read the rules first (see below), and note that we are not a catch-all for general images (of screenshots, comics, etc.)
a community for 6 yearsmessage the moderatorsMODERATORSqgyh2krispykrackersBritishEnglishPoliceTbluejamt9000nullrenGuitarFreak027Lynda73roger_roger_bot...and 10 more »234Mike Tyson's deserted mansion, complete with wall to wall zebra carpet and an ice cream sundae bar! (retardzone.com)submitted 1 year ago by LarryFlyntstone2 commentssharecancelsorry, this has been archived and can no longer be voted onloading...all 2 commentssorted by: hotbesttopnewhotcontroversialoldrandom[–]Goshawk3118191 1 point2 points3 points 1 year ago (1 child)sorry, this has been archived and can no longer be voted onHoly hell that is a lot of zebra carpet. Also, not a single squatter? Really? I find that hard to believe...
permalink[–]LarryFlyntstone[S] 0 points1 point2 points 1 year ago (0 children)sorry, this has been archived and can no longer be voted onIt's honestly in the middle of nowhere with farms all around it.
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Attack of the Killer Prom Dresses by Donna McClaire | 9781934614068 | NOOK Book (eBook) | Barnes & Noble
Attack of the Killer Prom Dresses
Donna McClaire
A feisty teenaged girl is ambitious for a career in dress design. Her fancy new sewing machine turns out to be controlled by aliens! One by one, from their lair in her closet, the aliens take control of her friends. Our determined heroine and her devoted boyfriend must block the aliens from their dire designs.
Attack of the Killer Prom Dresses available in
9781934614068
As townspeople walked briskly past the weird door she'd just come out of, Kim Howell twisted around behind her red Toyota's steering wheel and watched Mike stuff the huge TV-sized box into the back seat.She studied his face. Curly dark brown hair, large brown eyes under bushy brows, a straight nose with a little bitty bump in the middle, full lips. Luscious lips. She'd felt them on hers the first time the previous fall, after the Ft. Branch basketball game. He'd come out of the locker room where she was talking with Ashley and Gina, and they looked at each other and--well, she hadn't been the same since.Mike grunted and kneed the box up onto the seat. He looked real serious. That's probably what they meant when they said someone had "knitted brows," she figured. And boy, did he have the brows to knit. She leaned toward him and patted the box."Careful, there," she said. "You break this thing, my whole life's down the tubes."His eyes rolled up. "Right, right. Jeez, you'd think I was some kind of dweeb.""Not just 'some kind,' dahling," she said. "The very best kind."He grinned and pushed the box farther in. "I'll ride back here with it," he said. He jumped in beside the box and slammed the door.She glanced again at the strange shop doorway they'd just carried the box through. It was like a regular door up to waist high, then it got wider. The top part was shaped like a huge upside-down turnip pushed right into the brick wall. It looked like it was out of a mosque in the Far East or something, like she'd seen on the news. She shook her head and shifted into drive."I still don't remember that doorway," she said. "Eppe's drug store there onthe left, sure. The barber shop on the right, absolutely. They've probably been around ever since our Founding Fathers first camped here. But I sure don't remember that store. Do you?""Well, not really. But there it is.""Well, duh, Mikey, of course there it is. It's just that--well, somebody would remember a door like that."She shrugged and peeled out, then tapped the brakes to avoid a collision. She dramatically wiped her hand across her forehead, and said "whew" for effect. Downtown Bloomingdale was sure busy. Horns blasted, people shouted--you'd think you were in Chicago or something, not a little town in Indiana.She stopped at another light, and drummed her fingers on the steering wheel as she watched people go in and out of offices and stores. She usually shopped at Cordova Mall, south of town. She'd never even considered shopping on Main Street until she saw this unbelievable, super-deal Bernina sewing machine newspaper ad.The light changed and she floored it. Oops! Too late, she remembered the sewing machine. She stomped the brake. Mike bounced against the front seat."Kim!""Sorry. The machine okay?""Let me move this broken leg out of the way and check it," he said.She smiled. He was a funny guy, that Mikey. She turned right on Spring Avenue and slowed to make it through the timed lights. The traffic thinned and soon she was in the residential area, driving past dark green maples and an occasional red-splashed mimosa. Beech, Oak, Pine streets slipped by. She turned right at the next street, touched the brake, and glided to a stop in the driveway at 1410 Juniper. The two-story white frame house sat comfortably back on a trim lawn. She jumped out of the car and opened the back door."Careful, Mike. That's got to get me through three dresses in the next two weeks."Mike backed out of the car, dragging the box onto the ground."They could've put handles on it," he said. "Here--grab the other end."They picked up the box. She studied his face as he backed along the sidewalk toward the porch, looking over his shoulder. He glanced at the box to adjust his grip and--"Watch out!" she said.Too late. He missed the first porch step and the heavy load squished him against the railing. He landed on his rear end, and the box landed on him.He grinned. "Saved the Machine," he said in his best science fiction voice. "A thousand died, but the Force lives on.""A thousand and one, you keep that up. Hurry--we've got to get back to school."They lugged the box up the stairs to her bedroom, and dumped it onto a card table. He glanced around."Ah, heaven," he said. "The royal dressing table, the royal bed. The royal chest of drawers, the royal bed. The royal mirror, the royal bed--""And you're a royal pain, Mike." But she smiled as she said it. He'd called her his Queen Bee ever since she designed the dress with the black and yellow horizontal stripes. After that, everything was royal. Okay, okay, it wasn't her best work. But she was still learning, right?She glanced into the royal mirror. Nope. She didn't look like a bee, royal or otherwise. Bees didn't have black hair, black eyes, dark olive skin or dimpled chins. Or drop-dead figures, like Mike said she had. 'Nuff of that queen bee stuff, Mikey-boy, you got a real good deal here."Help me unwrap it," she said, with as much authority as she could muster. They tore at the Scotch tape and pulled the brown paper away, revealing a box covered with indecipherable foreign squiggles. She tore the box top open and Mike lifted the machine out. Its chrome trim sparkled."It says Berdina," he said, reading the one English word on the machine's side. He sounded disappointed. "Berdina with a "d." Sounds like Bernina, doesn't it?""That's what I thought it was," she said. "Bernina, not Berdina. I'm sure that's what the ad said. No wonder it was so cheap.""I guess I still don't know what's wrong with your mom's sewing machine." He pointed to the scratched up thing on the floor. "Looks perfectly good to me.""But all that one does is sew, Mike."He stared at her."I mean--it doesn't do anything fancy. Like make buttonholes, things like that. It just sits there and sews straight lines. I need more than straight lines if I'm going to win that scholarship.""Oh, right."He picked up some plastic peanuts that had dropped on the floor, and she sighed. He loved her, she knew. But he could never in his whole life understand her devotion to designing dresses. She'd made clothes for her dolls, graduated to simple things for herself. Then she got into it real big. She took night sewing classes at Bloomingdale Community College, even though she was just a high school Junior. She subscribed to sewing magazines, and took out every library book that had anything at all to do with dress design.And then the Albertson Fashion Design School in New York announced its scholarship contest. She'd read about it, and qualified to try for one of the five scholarships. Five in the whole nation! Mike said it was like his Bloomingdale Raiders making the state basketball finals.Mike put the peanuts into the box and shoved it into the closet. "Take in a movie tonight?""Can't. Got to meet the girls at the fabric store. Two weeks, Mikey Sweetbuns. Sure your dad doesn't mind doing the pictures?""I blackmailed him," Mike said. "He's downright eager."She hugged him and headed for the hall door. His father, a professional freelance photographer, did assignments for big-time ad agencies and magazines. Mike was learning the business, too.She glanced back at the sewing machine, sitting there next to the prom dress sketches. She'd been so sure it was a Bernina. She shrugged and they went out to the car. Mike slid into the passenger seat and she backed out of the driveway.Then she remembered the newspaper page she'd torn out, now laying crumpled in the back seat where Mike had sat on it. "Check the ad, Mike," she said. "I know it says Bernina.""Right."He got the paper, and she squeezed his leg. He was a good guy. Now she could sew those dresses for Ashley, Gina, and Sharon. Mr. Thompson would take the pictures on prom day, the girls would go to the dance, and she'd send the pictures and sketches to the contest headquarters. It was a great plan, actually.Mike frowned at the newspaper. "We got a little problem here," he said."What do you mean? It says Berdina?""No.""Well? Well, what?"He was quiet."Well, what?""It doesn't say anything."She waited for the punch line. "What do you mean?" she said, finally."We both read the ad, right? Took it into the store with us, even. Right?""Right.""Well, it's--the ad's disappeared right out of the newspaper!"There he goes. Teasing again.She glanced at him, ready for a real zinger of a come-back.But wait. He was serious. She'd seen that look before.Something was going on.No, Toto, we're not in Kansas anymore. A shiver went all the way up and down her spine.

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Patent US5217027 - Temporary cardiac lead - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsA temporary cardiac lead for establishing electrical contact between a body tissue and a pulse generator. The lead includes a flexible conductor having a proximal end and a distal end. The proximal end of the conductor is connected to the pulse generator. An electrode is connected to the distal end of...http://www.google.com/patents/US5217027?utm_source=gb-gplus-sharePatent US5217027 - Temporary cardiac leadAdvanced Patent SearchPublication numberUS5217027 APublication typeGrantApplication numberUS 07/707,722Publication dateJun 8, 1993Filing dateMay 30, 1991Priority dateMay 30, 1991Fee statusPaidAlso published asCA2102308A1, DE69205980D1, DE69205980T2, EP0588927A1, EP0588927B1, WO1992021406A1Publication number07707722, 707722, US 5217027 A, US 5217027A, US-A-5217027, US5217027 A, US5217027AInventorsMartinus A. J. M. HermensOriginal AssigneeMedtronic, Inc.Export CitationBiBTeX, EndNote, RefManPatent Citations (9), Referenced by (43), Classifications (5), Legal Events (5) External Links: USPTO, USPTO Assignment, EspacenetTemporary cardiac lead
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