Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

| SophiaSophia | 0 Comment
Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

The obligate intracellular bacterium Chlamydia psittaci is a recognized avian pathogen inflicting psittacosis in birds and is able to zoonotic transmission. In human pulmonary infections, C. psittaci could cause pneumonia related to important mortality if inadequately recognized and handled. Though intracellular C. psittaci manipulates host cell organelles for its replication and survival, it has been troublesome to show host-pathogen interactions in C. psittaci an infection as a result of lack of easy-to-handle genetic manipulation instruments. Right here, we present the genetic transformation of C. psittaci utilizing a plasmid shuttle vector that accommodates a controllable gene induction system.

The 7,553-bp plasmid p01DC12 was ready from the nonavian C. psittaci pressure 01DC12. We constructed the shuttle vector pCps-Tet-mCherry utilizing the complete sequence of p01DC12 and the 4,449-bp fragment of Chlamydia trachomatis shuttle vector pBOMB4-Tet-mCherry. pCps-Tet-mCherry contains genes encoding the inexperienced fluorescent protein (GFP), mCherry, and ampicillin resistance (AmpR). Goal genes may be inserted at a a number of cloning website (MCS). Importantly, these genes may be regulated by a tetracycline-inducible (tet) promoter.

Utilizing the pCps-Tet-mCherry plasmid shuttle vector, we present the expression of GFP, in addition to the induction of mCherry expression, in C. psittaci pressure 02DC15, which belongs to the avian C. psittaci 6BC clade. Moreover, we demonstrated that pCps-Tet-mCherry was stably retained in C. psittaci transformants. Thus, our C. psittaci plasmid shuttle vector system represents a novel focused strategy that permits the elucidation of host-pathogen interactions. The appliance of the pCps-Tet-mCherry shuttle vector system allows a promising new strategy to research unknown gene features of this pathogen.

Psittacosis, brought on by avian C. psittaci, has a serious financial influence within the poultry business worldwide and represents a big threat for zoonotic transmission to people. Prior to now decade, the instruments of genetic manipulation have been improved for chlamydial molecular research. Whereas a number of genetic instruments have been primarily developed in Chlamydia trachomatis, a steady gene-inducible shuttle vector system has to not date been out there for C. psittaci On this examine, we tailored a C. trachomatis plasmid shuttle vector system to C. psittaci We constructed a C. psittaci plasmid spine shuttle vector referred to as pCps-Tet-mCherry. The assemble expresses GFP in C. psittaci Importantly, exogeneous genes may be inserted at an MCS and are regulated by a tet promoter.

Detection of A number of Transgene Fragments in a Mouse Mannequin of Gene Doping Primarily based on Plasmid Vector Utilizing TaqMan-qPCR Assay

The World Anti-Doping Company has prohibited gene doping within the context of progress in gene remedy. There’s a threat that the augmentation of genes utilizing plasmids could possibly be utilized for gene doping. Nevertheless, no gold customary technique to detect this has been established. Right here, we aimed to develop a way to detect a number of transgene fragments as proof of gene doping. Firstly, gene supply mannequin mice as a mimic of gene doping had been created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the belly cavity. The outcomes confirmed profitable institution of the mannequin, with enough luminescence upon in vivo imaging.

Subsequent, a number of transgene fragments within the mannequin had been detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA utilizing the TaqMan- quantitative real-time PCR(qPCR) assay, with the best ranges in plasma cfDNA. Utilizing only a single drop of entire blood from the mannequin, we additionally tried long-term detection. The outcomes confirmed that a number of transgene fragments had been detected till 11 days. These findings point out that the mix of plasma cfDNA or only one drop of entire blood with TaqMan-qPCR assay is possible to detect plasmid-PEI-based gene doping. Our findings might speed up the event of strategies for detecting gene doping in people.

With the rise of recent highly effective genome engineering applied sciences, reminiscent of CRISPR/Cas9, cell fashions may be engineered successfully to speed up primary and illness analysis. Probably the most crucial step on this process is the environment friendly supply of overseas nucleic acids into cells by mobile transfection. Because the vectors encoding the elements essential for CRISPR/Cas genome engineering are all the time giant (9-19 kb), they end in low transfection effectivity and cell viability, and thus subsequent choice or purification of optimistic cells is required.

To beat these obstacles, we right here present a non-toxic and non-viral supply technique that will increase transfection effectivity (as much as 40-fold) and cell viability (as much as 6-fold) in plenty of hard-to-transfect human most cancers cell strains and first blood cells. At its core, the approach is predicated on including exogenous small plasmids of an outlined dimension to the transfection combination.

Development of a Plasmid Shuttle Vector System for Genetic Manipulation of Chlamydia psittaci

The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene supply vector

Non-viral gene supply methods have nice potential for protected and environment friendly gene remedy, whereas inefficient mobile and nuclear uptake stay as the key hurdles. Novel approaches are wanted to boost the transfection effectivity of non-viral vectors. In accordance with this want, the target of this examine was to assemble a non-viral vector that would obtain gene supply with out utilizing further lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by utilizing the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding inexperienced fluorescent protein (GFP) was labeled with quantum dots (QDs) by way of peptide-nucleic acid (PNA) recognition website.

Recombinant YopM protein was then connected to the conjugate by way of a second PNA recognition website. The YopM ̶ QDs ̶ pDNA conjugate was transfected into HeLa cells with out utilizing further transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was efficiently delivered to the nucleus. As management, bare pDNA was transfected into the cells by utilizing a business transfection reagent.

The egg drop syndrome (76) PCR kit
PCR-V044-96D 100T
EUR 686.4

Egg Drop Syndrome One-Step PCR kit
Oneq-V026-100D 100T
EUR 1039.2

Egg Drop Syndrome One-Step PCR kit
Oneq-V026-150D 150T
EUR 1177.2

Egg Drop Syndrome One-Step PCR kit
Oneq-V026-50D 50T
EUR 861.6

The egg drop syndrome (76) RT PCR kit
RTq-V044-100D 100T
EUR 860.4

The egg drop syndrome (76) RT PCR kit
RTq-V044-150D 150T
EUR 969.6

The egg drop syndrome (76) RT PCR kit
RTq-V044-50D 50T
EUR 717.6

The egg drop syndrome (76) One-Step PCR kit
Oneq-V044-100D 100T
EUR 1039.2

The egg drop syndrome (76) One-Step PCR kit
Oneq-V044-150D 150T
EUR 1177.2

The egg drop syndrome (76) One-Step PCR kit
Oneq-V044-50D 50T
EUR 861.6

PCR Mix
L5051100 2.5 ml
EUR 67

PRICE DROP
110-012 4 x 1000 µl
EUR 159.6

PRICE DROP
110-012L 5x 4 x 1000 µl
EUR 621.6

PRICE DROP
110-012XL 10x 4 x 1000 µl
EUR 1126.8

Ready? PCR Mix
M1127-1000 each
EUR 614.4

Ready? PCR Mix
M1127-200 each
EUR 229.2

Rigor? PCR Mix
M1132-200 each
EUR 385.2

Breeze? PCR Mix
M1134-200 each
EUR 320.4

Distant? PCR Mix
M1136-200 each
EUR 352.8

Advance? PCR Mix
M1139-200 each
EUR 451.2

Fire Start? PCR Mix
M1141-200 each
EUR 451.2

Ready? PCR Mix-Dye
M1128-1000 each
EUR 614.4

Ready? PCR Mix-Dye
M1128-200 each
EUR 229.2

Image Ready? PCR Mix
M1129-200 each
EUR 352.8

Rigor? PCR Mix-Dye
M1133-200 each
EUR 385.2

Whole Blood PCR Mix
M1143-200 each
EUR 385.2

Robust Ready? PCR Mix
M1130-1000 each
EUR 738

Robust Ready? PCR Mix
M1130-200 each
EUR 255.6

Breeze? PCR Mix-Dye
M1135-200 each
EUR 320.4

Distant? PCR Mix-Dye
M1137-200 each
EUR 352.8

Image Distant? PCR Mix
M1138-200 each
EUR 385.2

Advance? PCR Mix-Dye
M1140-200 each
EUR 451.2

PCR-EZ D-PCR MASTER MIX
BS294 100RXN, 100prep
EUR 112.2

amfiSure PCR Master Mix
P0311-010 2x50 rxns
EUR 151.2

amfiSure PCR Master Mix
P0311-025 5X50 rxns
EUR 198

amfiSure PCR Master Mix
P0311-050 10X50 rxns
EUR 262.8

amfiSure PCR Master Mix
P0311-100 10x1ml
EUR 343.2

amfiSure PCR Master Mix
P0311-125 15X50 rxns
EUR 333.6

amfiSure PCR Master Mix
P0311-200 20x1ml
EUR 562.8

amfiSure PCR Master Mix
P0311-250 50x50 rxns
EUR 902.4

amfiSure PCR Master Mix
P0311-500 100x50 rxns
EUR 1509.6

amfiXpand PCR Master Mix
P0331-010 2X50 rxns
EUR 181.2

amfiXpand PCR Master Mix
P0331-025 5X50 rxns
EUR 327.6

amfiXpand PCR Master Mix
P0331-050 10X50 rxns
EUR 537.6

amfiXpand PCR Master Mix
P0331-250 50x50 rxns
EUR 2247.6

Taq 2x PCR Mix 100 rxn
BA01501 100rxn
EUR 112.8
Description: High quality Taq polymerase for different PCR variations and downstream applications.

MyTaq Blood PCR Mix, 2x
BIO-25053/S Sample Ask for price

MyTaq Blood PCR Mix, 2x
BIO-25054 250 Reactions Ask for price

HotTaq 2x PCR Mix 100 rxn
BA01503 100rxn
EUR 144
Description: High quality HotTaq polymerase for different PCR variations and downstream applications.

RedTaq 2x PCR Mix 100 rxn
BA01507 100rxn
EUR 175.2
Description: High quality RedTaq polymerase for different PCR variations and downstream applications.

Fire Start? PCR Mix-Dye
M1142-200 each
EUR 451.2

HiScript-TS 2 × PCR Mix
RA103-01 40 rxns (25 μl/rxn)
EUR 69.6

HiScript-TS 2 × PCR Mix
RA103-02 200 rxns (25 μl/rxn)
EUR 275.2

Funnel Pyrex Cyl Drop Gr.500ml
FUN4024 EACH
EUR 176.4

Robust Ready? PCR Mix-Dye
M1131-1000 each
EUR 738

Robust Ready? PCR Mix-Dye
M1131-200 each
EUR 255.6

mRNAExpress Tail PCR mix (5uM)
MR-TAIL-PR 100 ul
EUR 111

Drop Delay Calibration Particles
DDCP-70-2 2 mL
EUR 339.6
Description: Please reffer to the technical data sheet for more detail information for this item. Our dedicated team would be happy to assist you via live chat, email or phone.

Drop Delay Calibration Particles
DDCP-70-20 20 mL
EUR 2301.6
Description: Please reffer to the technical data sheet for more detail information for this item. Our dedicated team would be happy to assist you via live chat, email or phone.

Taq 5x PCR Mix 12.5 mM
BT10402 250rxn
EUR 130.8
Description: High quality Taq polymerase for different PCR variations and downstream applications.

Drop Funnel Rotaflo SC 50ml
QD312 PK5
EUR 637.2

amfiSure Prime PCR Master Mix
P1311-025 5X50 rxns
EUR 193.2

amfiSure Prime PCR Master Mix
P1311-050 10X50 rxns
EUR 262.8

amfiSure Prime PCR Master Mix
P1311-125 15X50 rxns
EUR 327.6

amfiSure Prime PCR Master Mix
P1311-250 50x50 rxns
EUR 886.8

amfiSure Prime PCR Master Mix
P1311-500 100x50 rxns
EUR 1485.6

amfiSure Advanced PCR Master Mix
P2311-025 5X50 rxns
EUR 198

amfiSure Advanced PCR Master Mix
P2311-050 10X50 rxns
EUR 267.6

amfiSure Advanced PCR Master Mix
P2311-125 15X50 rxns
EUR 338.4

amfiSure Advanced PCR Master Mix
P2311-250 50x50 rxns
EUR 902.4

amfiSure Advanced PCR Master Mix
P2311-500 100x50 rxns
EUR 1534.8

Funnel Pyrex Cyl Drop Gr 1Ltr
FUN4026 EACH
EUR 249.6

amfiSure ONE PCR Master Mix(2X)
P7000-005 5x1 ml
EUR 262.8

amfiSure ONE PCR Master Mix(2X)
P7000-010 10x1 ml
EUR 380.4

amfiSure ONE PCR Master Mix(2X)
P7000-050 50x1 ml
EUR 1453.2

amfiSure ONE PCR Master Mix(2X)
P7000-100 100x1 ml
EUR 2522.4

Drop Funnel Cyl Rota S/C 100ml
QD1/21GP EACH
EUR 109.2

Drop Funnel Cyl Rota S/C 100ml
QD1/22GP EACH
EUR 116.4

Drop Funnel Cyl Rota S/C 250ml
QD1/32GP EACH
EUR 166.8

Drop Funnel Cylind Rota S/C 50ml
QD1/11GP EACH
EUR 102

Watershed Hanging Drop Cover Slip Endstation
M-WS180320-24WCSES 1 UNIT
EUR 256
Description: Watershed Hanging Drop Cover Slip Endstation

Taq PCR Master Mix (2X, Red Dye)
BS9297 1ml
EUR 101.76

Taq PCR Master Mix (2X, Red Dye)
BS9298 5ml
EUR 227.04

Taq PCR Master Mix (2X, Blue Dye)
BS9295 1ml
EUR 101.76

Taq PCR Master Mix (2X, Blue Dye)
BS9296 5ml
EUR 227.04

2×Taq PCR Master Mix(with dye)
K1034-1 1 ml
EUR 87.6

2×Taq PCR Master Mix(with dye)
K1034-100 100×1 ml
EUR 950.4

2×Taq PCR Master Mix(with dye)
K1034-20 20×1 ml
EUR 338.4

2×Taq PCR Master Mix(with dye)
K1034-5 5×1 ml
EUR 142.8

2×Taq PCR Master Mix(with dye)
K1034-50 50×1 ml
EUR 616.8

Drop Funnel P/Equal R S/C 100ml
QDE100/22 EACH
EUR 192

HotStart PCR Master mix (2X, Green Dye)
BS9291 5X1mL, 5ml
EUR 251.4

HotStart PCR Master mix (2X, Green Dye)
BS9292 1ml
EUR 115.68

amfiSure Ultra Fidelity PCR Master Mix(2X)
P0346-001 1ml
EUR 336

amfiSure Ultra Fidelity PCR Master Mix(2X)
P0346-002 2x1ml
EUR 578.4

amfiSure Ultra Fidelity PCR Master Mix(2X)
P0346-005 5x1ml
EUR 993.6

amfiSure Ultra Fidelity PCR Master Mix(2X)
P0346-010 10x1ml
EUR 1632

amfiFusion High Fidelity PCR Master Mix(2X)
P0342-010 2x50 rxns
EUR 303.6

amfiFusion High Fidelity PCR Master Mix(2X)
P0342-025 5X50 rxns
EUR 546

amfiFusion High Fidelity PCR Master Mix(2X)
P0342-050 10X50 rxns
EUR 934.8

amfiFusion High Fidelity PCR Master Mix(2X)
P0342-100 20x50 rxns
EUR 1599.6

Reverse Transcriptase RT PCR Master Mix (5X)
MB1002-100reactions 100 reactions
EUR 616.8

Reverse Transcriptase RT PCR Master Mix (5X)
MB1002-200reactions 200 reactions
EUR 895.2

Reverse Transcriptase RT PCR Master Mix (5X)
MB1002-25reactions 25 reactions
EUR 226.8

Extra T7 gRNA PCR primer mix (5 uM)
CAS510A-PR 50 assays
EUR 99

Taq 5x PCR Mix Ready-to-Load 12.5 mM
BT10502 250rxn
EUR 136.8
Description: High quality Taq polymerase for different PCR variations and downstream applications.

ReadiUseâ„¢ dNTP Mix Set *10 mM PCR Grade*
17258-1mL 1 mL
EUR 79
Description: 10 mM dNTP Mix is a mixture of four nucleotides (dATP, dCTP, dGTP, dTTP) in purified water.

Original INTELLI-PLATE 96-2; 2-well sitting drop, 120 plates
MAR-102-0011-00 120 PLATES
EUR 990
Description: Original INTELLI-PLATE 96-2; 2-well sitting drop, 120 plates

2x Genotyping PCR Ready Master Mix (250 x 20µL rxn)
T403 - Ask for price

INTELLI-PLATE 96-2 flat bottom; 2-well sitting drop, 120 plates
MAR-102-0001-01 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-2 flat bottom; 2-well sitting drop, 120 plates

INTELLI-PLATE 96-2 low profile;  2-well sitting drop, 120 plates
MAR-102-0001-10 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-2 low profile;  2-well sitting drop, 120 plates

INTELLI-PLATE 96-3 low profile; 3-well sitting drop, 120 plates
MAR-102-0001-13 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-3 low profile; 3-well sitting drop, 120 plates

96 WELL SITTING DROP HIGH THROUGHPUT CRYSTALLOGRAPHY PLATE.
CP-AXYGEM-96-50 10/pk
EUR 548.4
Description: Crystallography Plates; Crystallography Plates - Axygen

INTELLI-PLATE 96-2 Shallow well;  2-well sitting drop, 120 plates
MAR-102-0001-20 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-2 Shallow well;  2-well sitting drop, 120 plates

ReadiUse™ dNTP Mix Set *10 mM PCR Grade*
17258 1 mL
EUR 79

24 Well, Hanging Drop Plate, Polystyrene, W/LID, 24/cs
M-662150 24 PLATES
EUR 117
Description: 24 Well, Hanging Drop Plate, Polystyrene, W/LID, 24/cs

INTELLI-PLATE 96-2 low vol. reservoir; 2-well sitting drop, 120 plates
MAR-102-0001-00 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-2 low vol. reservoir; 2-well sitting drop, 120 plates

INTELLI-PLATE 96-3 low vol. reservoir; 3-well sitting drop, 120 plates
MAR-102-0001-03 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-3 low vol. reservoir; 3-well sitting drop, 120 plates

Accuris High Fidelity PCR Master Mix, sample, 5 rxns
PR1001-HF-S 1 each
EUR 7.1

Carbolite drop down door ELF 116B laboratory chamber furnace 6L
FUR1200 EACH
EUR 2676

Carbolite drop down door ELF 114B laboratory chamber furnace 14L
FUR1202 EACH
EUR 2881.2

INTELLI-PLATE 96-2 shallow well, low profile;   2-well sitting drop, 120 plates
MAR-102-0001-21 120 PLATES
EUR 990
Description: INTELLI-PLATE 96-2 shallow well, low profile;   2-well sitting drop, 120 plates

2x GoldStar Best PCR Master Mix (with Dyes), 20 ul/rxn
W0655-1 - Ask for price

2x GoldStar Best PCR Master Mix (with Dyes), 20 ul/rxn
W0655-5 - Ask for price

lyophilised real-time PCR Master Mix for dual labeled probes
M-PCR-156L 960 reactions x 20 µl
EUR 1023
Description: lyophilised real-time PCR Master Mix for dual labeled probes

lyophilised real-time PCR Master Mix for dual labeled probes
M-PCR-156S 192 reactions x 20 µl
EUR 256
Description: lyophilised real-time PCR Master Mix for dual labeled probes

amfiEco PCR Master Mix,8-Strip RTU(2X), for 50ul reaction
P0704-096 96 rxns
EUR 174

Aroclor Mix 1
PCB-M1 1ML
EUR 31.2

Smart 2X PCR pre-mix Pfu (30ul)
9K-002-0038 30uL, 30uL
EUR 697.37

Drop-n-Stain CF594 Goat anti-mouse IgG (H+L), highly corss-adsorbed
20957 5mL
EUR 206.4
Description: Minimum order quantity: 1 unit of 5mL

Drop-n-Stain CF543 Goat anti-mouse IgG (H+L), highly cross-adsorbed
20969 5mL
EUR 206.4
Description: Minimum order quantity: 1 unit of 5mL

Drop-n-Stain CF594 Goat anti-rabbit IgG (H+L), highly cross-adsorbed
20955 5mL
EUR 206.4
Description: Minimum order quantity: 1 unit of 5mL

Drop-n-Stain CF488A Goat anti-mouse IgG (H+L), highly cross-adsorbed
20956 5mL
EUR 206.4
Description: Minimum order quantity: 1 unit of 5mL

The Y. pseudotuberculosis YopM-functionalized conjugate achieved the best GFP expression, in comparison with different two YopM proteins and the transfection reagent. To the very best of our data, YopM protein was used for the primary time in a non-viral gene supply vector.

Tags: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ,

Leave a Reply

Your email address will not be published. Required fields are marked *

Related Post