( The Lambda Z...)A New Lambda Vector for Mammalian Expression

The Lambda ZAP-CMV vector for efficient library construction and mammalian expression

Quinn Lu Tanya Hosfield Cherie Dewar Tim Sanchez Mike Kobrin
Stratagene

Stratagenes new lambda cloning vector, the Lambda ZAP -CMV vector,* contains the left and right arms of the Lambda ZAP II vector and the pCMV-Script -EX phagemid vector. High-efficiency cDNA libraries constructed in the Lambda ZAP-CMV vector can be converted to plasmid libraries by a simple in vivo excision procedure. This vector preserves the benefits of lambda library construction, while providing the convenience of plasmids for expression in mammalian cells and characterization of cloned inserts.

Stratagenes family of Lambda ZAP vectors is designed to simplify the construction of high-titer cDNA libraries and the characterization of inserted DNA. These vectors are distinguished by their capability to easily excise and recircularize cloned insert DNA from lambda phage. When either single clones or the entire lambda library is converted to a plasmid format by in vivo excision,^1-5 they combine the high-efficiency cloning of lambda vectors with the convenience of plasmid libraries for functional studies. This elegant procedure requires very little hands-on time and eliminates the need for subcloning procedures.

High-Level Expression in Mammalian Cells

Figure 1

Stratagenes new lambda vector, the Lambda ZAP-CMV vector, offers efficient library construction and, upon mass excision, high-level eukaryotic expression in the pCMV-Script-EX phagemid vector. The Lambda ZAP-CMV vector (Figure 1) contains lambda arms derived from the Lambda ZAP II vector, three unique restriction sites in the multiple cloning site, and the pCMV-Script-EX phagemid vector.

Figure 2

The pCMV-Script-EX vector contains 15 unique sites for cloning (Figure 2) and offers the same features as its parental pCMV-Script vector;⁶ gene expression driven by the CMV promoter for constitutive expression in a wide variety of cell lines;^### the SV40 polyadenylation site; and the neomycin-resistance gene under control of the prokaryotic b-lactamase promoter, to provide kanamycin resistance in bacteria, as well as the SV40 early promoter, to provide G418 resistance in mammalian cells. The pCMV-Script-EX vector varies from its parental pCMV-Script vector by a 29-bp sequence downstream of the f1 origin; however, this additional sequence is in a nonfunctional region of the vector. Thus, the new pCMV-Script-EX vector is functionally equivalent to the pCMV-Script vector.

The Lambda-ZAP CMV vector is available in kits of varying components to accommodate different research needs. The Lambda ZAP-CMV XR library construction kit includes the directional cDNA synthesis kit, the Lambda ZAP-CMV XR vector, Gigapack III Gold packaging extract, host strains, and helper phage. Lambda ZAP-CMV vector kits are available that include the Lambda ZAP-CMV vector predigested with either EcoR I only or EcoR I and Xho I, host strains, and helper phage. In addition, the Lambda ZAP-CMV vector is available in undigested form with host strains and helper phage.

cDNA Library Construction in the Lambda ZAP-CMV Vector

Figure 3

Libraries constructed in the Lambda ZAP-CMV vector are characterized by high titers and robust plaque size. We constructed a cDNA library in the EcoR I and Xho I sites of the Lambda ZAP-CMV vector using poly(A)+ RNA from human HeLa cells. This library contained approximately 3 x 10⁶ primary plaques. The phage titer of the amplified library was 2 x 10¹⁰ plaque-forming units (pfu)/ml. To assess the library quality (background level and insert size), we mass excised and selected phagemid colonies by antibiotic selection on kanamycin plates. Plasmid DNA was prepared from 12 randomly picked colonies, and the DNA was digested with either EcoR I and Xho I or Not I and Kpn I restriction enzymes. All 12 clones contained a cDNA insert, ranging in size from 0.5 kb to 2.5 kb, with an average size of 1.4 kb (Figure 3). The titer of the amplified library in the Lambda ZAP-CMV vector remained constant upon storage at 4C, -80C, and in the presence of 7% DMSO.

Luciferase Expression in the pCMV-Script -EX Vector

Figure 4

To demonstrate expression levels in the pCMV-Script-EX vector, we inserted the firefly luciferase gene into the BamH I site of the MCS. This same luciferase gene was inserted into the parental pCMV-Script vector. These two constructs were used for parallel transfections of Chinese hamster ovary (CHO) cells. Cell lysates were prepared and assayed for luciferase activity. In Figure 4, cells transfected with each construct show comparable luciferase activity. We also detected luciferase protein in both samples by Western blot analysis (data not shown). To further demonstrate the functionality of the pCMV-Script-EX vector, we transfected the pCMV-Script-EX vector with luciferase gene construct into CHO cells and selected cells with G418 to establish a stable transfected cell line with high levels of luciferase expression (data not shown). These data confirm that the pCMV-Script-EX phagemid vector expresses high levels of protein in mammalian cells.

Lambda ZAP Vectors for Diverse Applications

Table 1
Stratagenes Lambda ZAP cDNA Library Vectors

Comparison of Lambda cDNA Library Vectors

HybriZAP 2.1
two-hybrid vector

ZAP Express
vector

Lambda ZAP-CMV
vector

Lambda ZAP II
vector

# unique cloning sites in l
(# sites in excised plasmid)

2
(7 in pAD-GAL4-2.1)

12
(17 in pBK-CMV)

3
(15 in pCMV-Script)

6
(21 in pBluescript SK- phagemid)

cloning capacity

6 kb

12 kb

6.5 kb

10 kb

eukaryotic expression

yes
(p_ADH1 promoter)

yes
(CMV promoter)

yes
(CMV promoter)

no

prokaryotic blue/white color screening
and b-gal fusion protein expression

no

yes

no

yes

contains excisable phagemid

yes

yes

yes

yes

antibiotic resistance

Amp^r

Kan^r

Kan^r

Amp^r

eukaryotic selection

leucine

G418

G418

none

directional cloning

yes

yes

yes

yes

suitable helper phage

VCSM13,
ExAssist helper phage

R408, RE704,
ExAssist helper phage

R408, RE704,
ExAssist helper phage

R408, VCSM13, RE704,
ExAssist helper phage

ssDNA mutagenesis

yes

yes

yes

yes

T3/T7 RNA polymerase promoters

T7 only

yes

yes

yes

exo-mung deletions

no

yes

yes

yes

The Lambda ZAP vectors (Table 1) are available for prokaryotic expression (the Lambda ZAP II vector), two-hybrid screening in yeast (the HybriZAP 2.1 vector), both prokaryotic and eukaryotic expression (the ZAP Express vector), and now, the new Lambda ZAP-CMV vector. The Lambda ZAP II vector is the most widely used vector for constructing cDNA libraries. Libraries cloned in this vector can be functionally screened with nucleic acid probes or antibodies. Alternatively, the library can be quickly and easily mass excised into the pBluescript SK(-) plasmid vector for analysis of transcripts via expressed sequence tag (EST) sequencing or subtraction and normalization procedures. The HybriZAP 2.1 vector offers the convenience of lambda library technology; after library construction, excision of the pAD-GAL4-2.1 plasmid vector allows cloned DNA to be screened for protein-protein interactions in vivo. The ZAP Express vector offers library construction with the capability for both prokaryotic and eukaryotic expression. Libraries constructed in the ZAP Express vector can be easily screened, and individual clones can be quickly converted to the pBK-CMV vector for eukaryotic transcription directed by the cytomegalovirus (CMV) immediate early promoter; this results in mid-level expression of cloned genes in mammalian cells.

Conclusions

Stratagenes Lambda ZAP vectors are designed for simplified construction of high-titer cDNA libraries, combined with the capability for excision and recircularization of cloned insert DNA from lambda phage. With the simple in vivo excision procedure, convert either individual clones or entire lambda libraries to a plasmid format. And, the newest lambda cloning vector, the Lambda ZAP-CMV mammalian expression vector, features the pCMV-Script-EX phagemid to achieve the highest levels of expression in mammalian cells.

Acknowledgments

We thank members of the Research and Development group, the Vector group, and the Custom Library group at Stratagene for discussion and suggestions.

PRODUCT LINK

REFERENCES

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2. Alting-Mees, M., et al. (1992) Strategies 5: 58-61.

3. Alting-Mees, M. and Short, J.M. (1989) Nucleic Acids Res. 17: 9494.

4. Amberg, J., et al. (1993) Strategies 6: 2-4.

5. Mullinax, R.L. and Sorge, J.A. (1995) Strategies 8: 3-5.

6. Hosfield, T., et al. (1997) Strategies 10: 68-69.

* U.S. Patent Nos. 5,128,256 and 5,286,636 and European Patent No. 286200