Description

Adenovirus Expression Vector Kit (Dual Version) Ver.2 is designed for making recombinant adenovirus producing by either of two methods; the full-length DNA transfer method or the COS-TPC method²⁾.
Dr. Saito et al. at the Institute of Medical Science at the University of Tokyo developed the full-length DNA transfer method⁴⁾ for simple production of recombinant adenovirus by constructing a dual cosmid³⁾ containing adenovirus DNA lacked E1 and E3 genes, which is a modified version of the cosmid vector for the COS-TPC method. The full-length DNA transfer method is for producing a target recombinant adenovirus by transduction only the plasmid containing full-length adenovirus into 293 cells.
This product contains the dual cosmid having BspT140 I and Pac I restriction sites outside of both terminal ends of virus genome. After a gene of interest is inserted in the dual cosmid, the recombinant adenovirus having the gene of interest is able to be obtained by transfection into 293 cells with the BspT140 I or Pac I-digested recombinant cosmid. In the kit Ver.2, you can select the restriction enzyme, BspT140 I or Pac I, which will be used for cutting the recombinant cosmid. Therefore, it is possible to produce the recombinant adenovirus having your gene of interest by the full-length DNA transfer method.
In a difficult case for production of a recombinant adenovirus using the full-length DNA transfer method based on sequence or function of a gene of interest, the COS-TPC method is available for production of recombinant adenovirus by co-transfection of the obtained recombinant cosmid together with Adenovirus genome DNA-TPC (Cat. #6171) into 293 cells. There is no difference in the structure or characteristics of recombinant viruses produced by both methods.

Storage

6170:

• 10% SDS: Thawed at 37°C and stored at room temperature.
• Others: –20°C

6171:

• Adenovirus genome DNA-TPC: –80°C

Feature

• Cost-efficient, economic kit with easy protocol
• Uses the Full Length DNA Transfer Method : Direct Cloning into a stable cosmid without use of a shuttle vector
• High efficiency of target clone production
• This kit contains all reagents(except DNA-TPC) to perform both Full length DNA Transfer method and COS-TPC method of cloning

Figure Principle of recombinant adenovirus construction

Overview of Procedure

1. 
   

   Insert a target gene into a cosmid vector.
   |
   |¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯|
   ↓	↓
   ♦ Full length DNA Transfer method	♦ COS-TPC method
   Digest the recombinant cosmid by restriction enzyme BspT104 I or Pac I and transfect it into 293 cells. Recombinant adenovirus is produced in the cell.	Co-infect DNA-TPC* and recombinant cosmid inserted with a target into 293 cells. Homologous recombination occurs within a cell and Recombinant adenovirus is produced.
   |−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−| ↓

4. Non-proliferating type of recombinant adenovirus proliferate, since 293 cells express E1A and E1B genes. The structure and nature of the obtained adenovirus is the same between the 2 methods.
5. Infect the constructed adenovirus into a target cell. In normal cells, the adenovirus does not proliferate, but express target protein.

Kit components

Adenovirus Expression Vector Kit (Dual Version) Ver.2

1. Cosmid Vector pAxcwit2*1 (0.3 µg/µl)	25 µl
2. Cosmid Vector pAxCAwtit2*2 (0.3 µg/µl)	25 µl
3. Smi I (Swa I) (10 U/µl)	20 µl
4. 10 × H Buffer	100 µl
5. BspT104 I (10 U/µl)	30 µl
6. 10 × L Buffer	100 µl
7. DNA Dissolution Buffer	50 µl
8. Ligation Solution	50 µl
9. 10 × TNE	1 ml × 2
10. Proteinase K (20 mg/ml)	200 µl
11. 10% SDS	200 µl
12. Control Cosmid pAxCAiLacZit*3(0.3 µg/µl)	50 µl

*1 With no promoter
*2 With CAG promoter
*3 pAxCAwtit with β-gal gene insertion

Structure of Cosmid Vectors

The Adenovirus Expression Vector Kit (Dual Version) Ver.2 contains two types of dual cosmids, pAxcwit and pAxCAwtit (Figures 2-1 and 2-2), both of which are cosmid vectors containing the entire adenovirus genome, except for the E1 and E3 genes.
• pAxcwit: a basic vector lacking a promoter sequence that allows for the insertion of up to about 7 kbp of foreign an expression cassette (promoter + target gene + poly (A) signal).
• pAxCAwtit: a vector containing CAG promoter (cytomegalovirus enhancer, chicken β-actin promoter, and rabbit β-globin poly(A) signal), which is a strong mammalian promoter, that allows for the insertion of up to about 5 kbp of a gene of interest.

Both vectors contain Smi I (Swa I) and Cla I cloning sites.
For high-level expression, clone the coding region of the gene of interest into the pAxCAwtit vector. To use a different promoter, clone an expression cassette (promoter + coding region of the gene of interest + poly(A) signal) into the pAxcwit vector.
The constructed recombinant cosmids can be used with both the full-length DNA insertion and the COS-TPC recombinant adenovirus preparation methods. The structures of the pAxcwit and pAxCAwtit vectors are shown in Figures 2-1 and 2-2.

*a: Sal I has the other restriction sites in this cosmid; at (5,189), (7,236), (9,283), (11,661), (29,260), (36,165), (36,544) *b: Nru I has the other restriction sites in this cosmid; at (3,779), (4,868), (5,826), (6,915), (7,873), (8,962), (9,664), (24,133), (28,416), (34,579), (34,660), (38,273), (39,692). *c: The site marked with (ClaI) are not digested with the enzyme because it is affected by dam methylation. Notes: The restriction sites of the above figure are shown with the 5′ end of the recognition site of the restriction enzymes except Smi I (SwaI) and BspT104 I. The sites of Smi I (SwaI) and BspT104 I are displayed with its cutting sites.

Fig.2-1 Structure of cosmid vector pAxcwit2
pAxcwit2 is a basic vector and does not contain a promoter sequence.
A foreign DNA of about 7 kbp can be inserted into Smi I or Cla I site.

*a: Xba I has the other restriction sites in this cosmid ; (14,224), (19,730), (37,734). *b: PshB II has the other restriction sites in this cosmid ; (25), (2,801), (3,660), (14,256), (14,781), (17,121), (17,469), (18,147), (18,471), (28,221), (28,836). *c: Xho I has the other restriction sites in this cosmid ; (23,527), (38,029), (38,624), (40,069),(42,535). *d: Bgl II has the other restriction sites in this cosmid ; (15,813), (16,085), (17,710), (19,382), (20,189), (23,133), (25,284), (26,552), (31,730), (37, 912), (39,409). *e: Sal I has the other restriction sites in this cosmid ; (7,506), (9,553), (1111,600), (13,978), (31,577), (38,482), (38,861). *f: Nru I has the other restriction sites in this cosmid ; (6,096), (7,185), (8,143), (9,232), (10,190), (11,279), (11,981), (26,450), (30,733), (36,896), (36,977), (40,590), (42,009). *g: Spe I has the other restriction sites in this cosmid ; (18), (21,241). *h: The site marked with (Cla I) are not digested with the enzyme because it is affected by dam methylation. *1: Cytomegalovirus enhancer + chicken β-actin promoter + a part of 3′ untranslated region of rabbit β-globin. *2: rabbit β-globin polyA Notes: The restriction sites of the above figure are shown with the 5′ end of the recognition site of the restriction enzymes except Smi I (Swa I) and BspT104 I. The sites of Smi I (Swa I) and BspT104 I are displayed with its cutting sites.

Fig.2-2 Structure of cosmid vector pAxCAwtit2
pAxCAwtit2 contains highly efficient CAG promoters (Cytomegalovirus enhancer, chicken β-actin promoter, rabbit β-globin polyA signal)²⁾ that act in mammals.
A foreign DNA of about 5 kbp can be inserted into Smi I or Cla I site.

Sequence of Cosmid Vectors

Sequence of pAxcwit2
Sequence of pAxCAwtit2
Sequence of pAxCAiLacZit

The Sequences of cosmid vectors are compressed into ZIP format. They are expanded with StuffIt Expander (for Macintosh), WinZip (for Windows).