Nucleic Acids Research, Vol. 19, No. 12 3403

k.) 1991 Oxford University Press

pXZd39: a new type of cDNA expression vector for low background, high efficiency directional cloning Andrew J.M.Murphy and Robert T.Schimke Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA Received January 28, 1991; Revised and Accepted May 10, 1991

ABSTRACT We have developed a new type of bacteriophage X vector which provides a strong biological selection against non-recombinants that is independent of the sequences immediately surrounding the cloning site. This system, which we call 'selective substitution', is ideally suited for cDNA expression vectors where it is necessary to flank the cDNA insert with sequence elements (promoters etc.) required to produce a biologically active mRNA in vivo. Selective substitution is a general method, which may be applied to many types of vectors. In this report, we have specifically applied selective substitution to the construction of a new mammalian retrovirus expression vector. The level of background obtained with this vector (that is, the number of plaques obtained when the vector is ligated in the absence of insert DNA) is 0.02% when compared to ligation with restriction fragments and 0.1% to 0.4% when compared to ligation with newly synthesized cDNA. These features have allowed us to easily and efficiently generate several large cDNA libraries using total and size selected cDNA.

INTRODUCTION Due to the large size of the mammalian genome and the broken intron/exon structure of most mammalian genes, cDNA cloning is usually an important step in the elucidation of the structure and function of mammalian genes. While cDNA cloning techniques had existed for some time (ref. 1, for example), the introduction of the cloning vector XgtlO (2) was a major technical advance owing to that vector's high efficiency, ease of use and inherent low background. Aside from the greater efficiencies and ease of storage of X over plasmid vectors, the unique advantages of XgtlO stem from the use of a single EcoRI cloning site within the cI repressor gene of its bacteriophage genome. Cloning into this site inactivates the repressor gene allowing recombinants to form plaques on a special E. coli strain in which nonrecombinants will not grow. This biological selection greatly reduces background while it alleviates the necessity for efficiencyreducing phosphatase treatment of the vector. Unfortunately, the XgtlO method of selection against non-recombinants by insertional inactivation is not conducive to the construction of expression vectors, since the interrupted cI gene sequences are left flanking the insert instead of the desired promoter and polyadenylation, or terminator sequences.

Thus, we have constructed a new X vector which uses 'selective substitution' instead of insertional innactivation as a mechanism to achieve a biological selection against non-recombinants. The system allows large cDNA libraries to be easily constructed in expression vectors with very low backgrounds. The system also allows directional cloning of cDNA, effectively doubling the number of expressible clones compared to non-directional libraries. Low backgrounds and directional cloning are especially important for expression cloning of rare cDNAs in mammalian systems where the efficiency of transfection into the cultured cells is the limiting step. Extremely low backgrounds are also important in cases where the cDNA to be cloned may be of poor quality or of low amounts and uncertain quantity, like after extensive size selection.

MATERIALS AND METHODS General cloning methods Packaging extracts. Packaging extracts were prepared from BHB2688 and BHB2690 by the modified method of Barbara Hohn as described (3) except that putrescine was omitted. Phage were plated on the E. coli strain K802. Ligations in the presence of PEG. Ligation reactions to produce X vectors and libraries were performed in the presence of 15 % PEG (4) for 1 hour at room temperature at DNA concentrations of 5 to 15 ng/ml. Ligation products were pelleted by microcentrifugation for 5 minutes, decanted, carefully resuspended in a small volume of TE (10 mM TrisCl pH 7.5, 1 mM EDTA) and allowed to sit at room temperature for 20 minutes before in vitro packaging. Some small-scale control ligations were not pelleted after the 1 hour ligation reaction. Instead, in these cases, ATP was added to the reaction to a final concentration of 5 mM and after 20 minutes at room temperature, a portion of the reaction was added directly to the packaging extracts. All ligations were performed with approximately equimolar X vector and insert DNAs. Plasmid preps. Plasmids were prepared by the LiCl/PEG procedure (5) essentially as described (6). In addition, pXZd39 to be used as cDNA cloning vector was banded once on CsCl. Potassium acetate gradients. For purification of arms and size fractionation of cDNA 5-20% potassium acetate gradients (3, 7) containing 50 mM Tris pH 8, 10 mM EDTA were formed

3404 Nucleic Acids Research, Vol. 19, No. 12 in SW41 tubes. Centrifugation was at 200 with the speed and times given below for each experiment. The potassium acetate solutions were filtered through 0.45 / filters before use.

Linearpolyacrylamide cater. Linear polyacrylamide carrier was prepared as follows. A 15 ml solution was prepared containing 5% polyacrylamide (no bis), 0.3 M sodium acetate pH7, approximately 50 mg of bromophenyl blue, approximately 50 mg of ammonium persulfate and 100 /1 of TEMED. The solution was incubated at 650 for 30 minutes to allow the acrylamide to polymerize. The solution became viscous but not solid. After cooling to room temperature, the linear polyacrylamide was precipitated by the addition of 30 ml of ethanol. The carrier was pelleted, resuspended and reprecipitated until the ethanol supernantant was no longer blue. The final pellet was dried and resuspended in 15 ml of water. The resultant solution is 5 % or 50 mg/ml of linear polyacrylamide carrier. The bromophenyl blue may act as a chain terminator of acrylamide polymerization as the longer size fractions (as assayed by sepharose 4B chromatography) are less blue than the shorter ones.

Construction of pXZd39 See RESULTS AND DISCUSSION for the rationale behind the following manipulations. XZd35B. XZd35 (8) DNA was ligated to protect the cohesive ends, digested with BamHI and end-filled using the Klenow fragment of E. coli pol I. The blunt-ended fragments were then religated in the presence of PEG and in vitro packaged. The resultant plaque purified phage, which was identical to XZd35 except that it lacked both BamHI sites, was digested with XbaI (one site in each LTR). The gel-purified arms were ligated to XbaI digested rpZd5 (8) and packaged in vitro. The derivative having a structure identical to XZd35, except that it contained a single BamHI site, was named XZd35B.

rpZd7 and rpZd8. rpZd5 was digested with KpnI (a single site in the LTR) and EcoRI and the gel-isolated large fragment was ligated to a 984 bp KpnI to EcoRI fragment derived from pLNL6 (9). The EcoRI site was eliminated from the resultant plasmid (rpZd6) by digesting, end-filling and religating to generate rpZd6ARI. This plasmid was then digested with BamHI, phosphatase treated and ligated to the tri-initiator oligonucleotide duplex (8). The plasmid with the tri-initiator in the productive orientation was designated rpZd7 while the plasmid containing the tri-initiator in the opposite orientation was called rpZd8. XZd37. After XZd35B DNA was digested with XbaI, the gelisolated arms were ligated to XbaI-linearized rpZd7 and in vitro packaged to generate XZd37. This X vector is similar to XZd35 except that it contains the extended retroviral packaging site from pLNL6 and a unique BamHI cloning site next to the EcoRI cloning site.

pX39. XZd37 DNA digested with BamHI and EcoRI was ligated to the approximately 1.7 kb BamHI to EcoRl fragment derived from pEA305 (10) which contains the lambda repressor gene under the control of the tac promoter. The ligation products were packaged in vitro and used to infect the lacIsq strain DH21 (11). The infected cells were allowed to express antibiotic resistance after phage absorbtion by adding 1 ml of broth and incubating at 370 for 1 hour. The cells were then spread onto plates containing 75 jg/ml ampicillin, 50 ,g/ml kanamycin and

20 mM Sodium Citrate. A colony was picked and found to contain a plasmid with the structure of pXZd39 which was subsequently purified away from contaminating XZd37 by calcium chloride transformation of DH21. Preparation of vector for cloning A culture of pXZd39 in DH21 was grown in media containing 75 jug/ml ampicillin, 50 yg/ml kanamycin and 20 mM Sodium Citrate. Plasmid was purified and 50 ,tg was digested with BamHI and EcoRI. After phenol extraction, half of the reaction mixture was layered onto each of two potassium acetate gradients and centrifuged at 27,000 rpm for 15 hr. The vector pellets under these conditions while the X repressor containing stuffer fragment and any contaminating chromosomal fragments remain in the body of the gradient. The tubes were decanted by inversion and the excess liquid wiped off with a paper towel. The pelleted vector was then resuspended in TE and ethanol precipitated to remove traces of salt.

cDNA synthesis First strand synthesis. First strand cDNA synthesis was performed in a 100 itl reaction containing the buffer supplied by the manufacturer (BRL), 7 ,ug of polyA+ RNA isolated from mouse A9 cells, 5 Mg oligo dT12_18 (Pharmacia), 500 MM each of the four dNTPs (Pharmacia), 16 MCi of 32P dCTP (3000 Ci/mmole, Amersham) and 1600 units of Moloney reverse transcriptase (BRL). After 1 hour at 370C the reaction was terminated by the addition of EDTA to 25 mM and phenol extraction. Unincorporated nucleotides and oligo dT were removed by chromatography over a 5 ml G100 (Pharmacia) column pre-equilibrated in TE plus 300 mM sodium Acetate. First strand yield was determined by counting aliquots of the reaction before and after G100 chromatography. Tailing. After ethanol precipitation, the first strand cDNA was tailed in a 100 pl reaction containing the cDNA, 50 mM potassium cacodylate pH 7.2, 1 mM CoCl2, 0.1 mM DTT, 0.12 mM dGTP and 125 units of terminal transferase (Boehringer) at 370 for 15 minutes. The reaction was terminated by the addition of 5MA1 of 0.5 M EDTA, phenol extracted, ethanol precipitated and resuspended in 25 ML1 of TE.

Second strand synthesis. The G-tailed first strand cDNA was combined with 2 M1 of oligo dC12_18 (2 Mg'lM, Pharmacia) and 10 M1 of 10xnick salts (200 mM Tris pH 7.5, 100 mM (NH4)2SO4, 1 M KCl) and annealed at 450 for 10 minutes and 370 for 10 minutes and then placed on ice. The reaction was adjusted to 100 M1 final volume containing 4 mM MgCl2, 150 MtM ,B-NAD, 200 MLM each dNTPs, 32 MLCi of 32P dCTP, 5 units of RNAse H (Boehringer), 5 units of E. coli DNA ligase (New England Biolabs) and 20 units of E. coli DNA polymerase I (New England Biolabs) and incubated at 150 for 1 hour and 220 for 1 hour. The reaction was terminated, the double stranded cDNA chromatagraphed on G100 and the yield determined as for the first strand.

Methylation and linker addition. The double stranded cDNA was methylated in a 100 pil reaction containing 10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA, 0. 1 mM DTT, 80 MM S-adenosyl methionine, 32 units of BamHI methylase (New England Biolabs) and 40 units of EcoRI methylase (New England Biolabs). After phenol extraction and ethanol precipitation, the methylated cDNA

,.

Nucleic Acids Research, Vol. 19, No. 12 3405

antibiotic resistance genes reside elsewhere within the vector. Cleavage of the large plasmid with BamHIl and EcoRI excises the X repressor gene which is then physically separated from the vector 'arms' by pelleting the arms through a potassium acetate gradient under conditions whereby the repressor gene fragment stays in the body of the gradient. The isolated arms are then ligated to cDNA which has been constructed to contain an EcoRI 5' end and a BamHI 3' end (see cDNA cloning and size selection below). The ligation products are packaged in vitro into X phage particles which are, in turn, plated onto a lawn of bacteria. Phage from the resultant plaques are harvested and these constitute the final amplified library. Any vector DNA which is not cut with both BamHI and EcoRI can reform a DNA molecule which contains the tac-cI fragment instead of a cDNA insert. These molecules can be packaged but they will not form a plaque upon plating due to the action of the tac-cI gene, and thus, they will not contribute to the background of non-recombinants. Since EcoRI and BamHI cohesive ends can not be ligated to each other at any appreciable rate by T4 DNA ligase, the only potential contributions to background are from i.) extraneous contaminating endo- or exonucleases, which are minimal due to the high quality of modern commercial restriction enzymes and the fact that a large excess of enzyme is not required to digest the vector DNA by this technique or ii.) mutations within the tac-cI gene or operator sequences, which are selected against during the growth of the

was ligated to 3 ,ug of phosphorylated R5 linker (sequence = AATTCGGATCCGAATT) using T4 DNA ligase overnight at 150. The next day the ligation reaction was heat inactivated at 650 for 20 minutes and digested with 750 units of BamHI and 50 units of EcoRI. This reaction was then terminated with EDTA and phenol extraction. Vector-ready cDNA was generated after GIOO chromatography and ethanol precipitation.

Tissue culture Wgd5 and methods for retroviral transduction by transient expression / co-cultivation have been described (8). WOP cell are described in (12). PA317 is described in (13). Fao is described in (14). All cell lines were maintained in Dulbecco's Modified Eagle's Medium plus 10% fetal bovine serum and 50 jitg/ml gentamycin.

RESULTS AND DISCUSSION Description of the selective substitution method In the pXZd39 system (see Fig. 1), a biological selection is achieved by inserting a constitutively expressed version of the X cI repressor gene between asymmetric BamHI and EcoRI cloning sites of the bacteriophage X derived retrovirus expression vector XZd37. The action of the repressor gene prevents pXZd39 from growing as a lytic X phage. However, it can be propagated as a large plasmid, because plasmid replication origins and SV40

LTR

Rl ,Bam

neo/kan

Rl

,

tac

LTR

ori pBR ori

.Zd37 (44.5 kb)

Bam

tac-cl

Rl

\

E

+

~

'B } insert to generate pUZd39

cl

0_

promoter

1Bam

/ am

(

pXZd39 -46 kb

Digest with Rl and Bamr cos

R

isolate vector and ligate to cDNA

Rl

Bam cDNA

package and amplify

l

l

l yyll

Figure 1. Schematic representation of vector construction and use. Not drawn to scale. The various steps in the construction and use of pXZd39 are described in the text. Bacteriophage X DNA, restriction fragments and concatamers are shown as linear. The pXZd39 plasmid is shown as a circle. The tac-cI fragment is stippled while cDNA is striped. RI = EcoRl, Bam = BamHI. LTR=long terminal repeat. ori=origin of replication.

3406 Nucleic Acids Research, Vol. 19, No. 12

pXZd39 since they would cause the plasmid to enter lytic growth and kill the cell. This latter is an advantage over even the XgtlO system, as repressor mutants of that phage form clear plaques which have a growth advantage over the wild type during its propagation.

Construction of pXZd39 from XZd35 pXZd39 was constructed from XZd35 (8) in three steps (see MATERIALS AND METHODS for details). First, a Bam site in the right X arm was removed leaving a unique Bam cloning site. Second, the extended retroviral packaging sequence from pLNL6 (9) was added. And third, an approximately 1.7 kb Bam to EcoRi fragment from pEA305 (10) containing the X cI repressor gene under the control of the tac promoter was inserted at the cDNA cloning site. The presence of this highly expressed X repressor gene in the construct effectively prevents its propagation as a phage (eliminating any background from uncut vector) while allowing it to be maintained as a plasmid. The tac promoter is so strong, however, that the large amount of repressor that is generated from this hybrid gene is toxic in wild-type cells (10). Thus, pXZd39 is grown in DH21 (11), a strain which overproduces the lac repressor 50 fold and, thus, down regulates the tac promoter. pXZd39 grows well in this host with typical yields being greater than 1 milligram of plasmid per liter of culture. Control ligations with cloned fragments As a first test of the cloning system, 100 ng of pXZd39 vector, prepared for cloning as described in MATERIALS AND METHODS, was ligated to an approximately equimolar amount of pBR322 cut with EcoRI and BamHI. Digestion of pBR322 with EcoRI and BamHI produces two fragments of 375 and 3988 base pairs, both of which possess one EcoRI end and one BamHI end. A second control ligation contained 100 ng of vector DNA, but no pBR322 DNA. One quarter of the ligation reactions were in vitro packaged, aliquots were plated onto E.coli lawns and plaques were counted the next day. As shown in Table 1, approximately 500 fold more plaques were obtained when pBR322 DNA was included in the ligation, a sizable increase over background. cDNA cloning and size selection Double stranded cDNA was synthesized from mouse A9 cell polyA+ RNA as described in MATERIALS AND METHODS

and as outlined in Fig. 2. This method of cDNA synthesis is a modification of published protocols (8, 15, 16, 17) which is highly efficient and produces cDNA with a high proportion of 5' ends (15) which can be directionally cloned. Starting with 7 p.g of polyA+ RNA, a yield of 1.2 ,tg of first strand cDNA was calculated by incorporation of radioactive nucleotide. Curiously, the yield of second strand cDNA was calculated to be 2 Ag, implying that either these yields were miscalculated (a possibility, since very small volumes were removed for scintillation counting) or 0.8 /tg of the second strand yield was derived from non-templated or 'rolling hairpin' synthesis. The yield of double stranded cDNA at this point was estimated to be 3.2 itg, an average between the first and second strand yields, but doubled to account for double-strandedness. After losses during the methylation and linker addition procedures, a final yield of 1.8,tg of double stranded cDNA with 5' EcoRI and 3' BamHI sites was obtained. A small portion of this (75 ng = 1/24 of the total) was ligated to 2 tsg of prepared pXZd39 vector, and the ligation products packaged in vitro. Small aliquots of the packaging reaction were plated and the plaques counted revealing that 1.44 x 106 independent clones had been obtained. This corresponds to an efficiency of 4.9 x 106 clones per jig of polyA+ RNA with a background of 0.3 % (Table 1). It is notable that this high cloning efficiency and low background was achieved without titration or very accurate quantitation of the input cDNA.

oligo dT, Reverse Transcriptase

dGTP, Terminal Transferase cap -

-

pfu/i4g of vector background

pfu/,ug of insert

insert pBR Bam+EcoRI total cDNA fractions 1-4 fractions 5-10

1.8x 103 9.9x 106 7.2x105 5.0x105 1.8 x 106

N/A 9.9x107 1.9x107 5.3x106 2.5 x 107

no

N/A 0.02% 0.3% 0.4% 0.1 %

A-A-kA.AA

oligo dC, pol CCCCCC GGGGGG

AAAAAA TTTTTT

Bam & EcoRI methylase GGGGGG

insert

-

TTTTTT

CCCCCC

Table 1. Cloning efficiencies of various inserts in pXZd39. The number of plaque forming units (pfu) after in vitro packaging were determined for each ligation by counting a representative dilution containing more than 50 plaques, except in the case of 'no insert' in which 10 ng of vector plated yielded 18 plaques. The backgrounds were determined by dividing the number of pfu/4g of vector for the control ligation (1.8 x 103) by the number of pfu/4g of vector for the test ligation. The mass of cDNA was estimated as described in the text for total (unfractionated) cDNA. The mass of cDNA in each size fraction was calculated as the ratio of radioactivity in that fraction to the total amount of radioactivity loaded multiplied by the total yield of cDNA. All ligations were performed using equimolar amounts of vector and insert, using the apparent average sizes of cDNA fractions to determine their molar amounts.

-

GGGGGG

CH3

AAAAAA TTTTTT

CH3

R5 oligo, T4 ligase CH3

AATTCGGATCCGAATTCCCCCC

TTAAGCCTAGGCTTAAGGGGGG-

AAAAAAAATTCGGATCCGAATT TTTTTTTTAAGCCTAGGCTTAA

CH3

EcoRt AATTCCCCCC GGGGGG

+ Bam

CAAAAAAAATTCG CH3

TTTTTTTTAAGCCTAG

size select or clone directly

Figure 2. Schematic representation of method of cDNA synthesis. The method of cDNA synthesis is fully described in the text. PolyA+ RNA is represented as a broken line. First and second strand cDNA is represented as a solid line. Poly A tails and oligonucleotide tracts are shown as shorter than actual length to save space. The 5' end of the RNA and the second (top) strand cDNA is on

the left, while the 5' end of the first (bottom) strand cDNA is on the right. Internal methylated nucleotides are represented as CH3.

Nucleic Acids Research, Vol. 19, No. 12 3407 The remainder of the cDNA was size fractionated by centrifugation over a potassium acetate gradient at 30,000 rpm for 17.5 hours. Fractions of 0.5 ml were withdrawn from the bottom of this gradient and precipitated at -20° for 30 minutes after the addition of 5 jig of linear polyacrylamide carrier and 1 ml of ethanol. A significant amount of radioactivity was also present, apparently pelleted, at the bottom of the gradient. This was resuspended and ethanol precipitated in parallel with the other fractions. Each fraction was resuspended in 50 Al of TE, and 5 pA1 of each of the first 17 (out of 22) fractions was electrophoresed on an agarose gel. The gel was fixed, dried and exposed to X-ray film (Fig. 3). An interesting feature of the gradient is the apparent partial aggregation of material in both the gradient and the gel with a size of about 2.8 kb (marked by arrows in Fig. 3). This material, which may represent the mysterious excess second strand synthesis described above, does not seem to be clonable since no stimulation above background was observed when the pellet fraction was ligated to pXZd39 vector.

The remainder of fractions 1 through 4 (95 ng of cDNA) were pooled and ligated to 1 ,ug of prepared pXZd39 vector to generate a sub-library of 5 x 105 clones with a calculated background of 0.4% (Table 1). The remainder of fractions 5 through 7 (177 ng of cDNA) were pooled and ligated to 2.5 p.g of prepared pXZd39 vector to generate a second sub-library of 4.45 x 106 clones with a calculated background of only 0.1 % (see Table 1). Analysis of cDNA length The total A9 library and the size selected library derived from fractions 1 through 4 were amplified and 'released' by

bottom P

1

2

3

4

5

m

6

7

8

9

10

11

12

13 14

m

15

-5 -4

-5 -4 -3 -

00 -2

I

~~~~~-2

s=

-1

*

-.s

-.5 _

..

Figure 3. Size fraction of cDNA over a potassium acetate gradient. Double stranded cDNA was size fractionated on a potassium acetate gradient as described in the text. Twenty-two 0.5 ml fractions were collected from a 20 guage syringe needle punctured into the bottom of the gradient. Material which had pelleted to the bottom of the gradient was also resuspended. A portion of each of the bottom 17 fractions (marked 1 through 17) were electrophoresed on an agarose gel along with a portion of the pellet (marked P) and cold size markers (1 kb ladder, BRL; marked m). The positions of the size markers were marked by boring a hole in the gel at the appropriate position with a pasteur pipet. The gel was acid fixed, dried onto filter paper and exposed to X-ray film for 3 hours with an intensifying screen at -80° along with dots of radioactive ink. The positions of the size markers were transfered by aligning the 'hot dots' on the gel with their images on the autoradiogram. The arrows mark material of apparent mobility of 2.8 kb which seems to aggregate in both the gradient and gel.

homologous recombination in vivo (8). A small portion of the released library DNA (about 1 ng) was transformed into bacteria and several random colonies (10 from the total library and 20 from the size selected library) were picked and analyzed as to the presence and sizes of cDNA inserts by digestion of miniprep DNAs with EcoRI and BamHI. This analysis (not shown) revealed that 1 of the clones from the total library and 4 clones from the size selected library did not have inserts. However, with one exception, these insertless clones did not have the right sized vector band, suggesting either that they are aberrant release products or that they have lost one of the restriction sites during cloning. This was confirmed by picking 35 separate plaques from the amplified library and isolating plasmid DNA from each using a scaled down release protocol. This analysis (not shown) revealed that 34 of the 35 clones had inserts which were excisable with BamHI and EcoRI. The remaining clone had apparently lost one of its cloning sites as it was linearized by BamHI plus EcoRI digestion to a size larger than vector alone. In addition, we have recently developed a new non-retroviral cloning vector in which a more efficient release mechanism utilizing site specific recombination has been employed along with selective substitution, and every one of the greater than 50 independent isolates from a library constructed in this new vector have contained inserts (Murphy et al., manuscript in preparation). The average size of inserts from the total A9 library was 550 bp, with the largest being 950 bp, whereas the average size of inserts from the size selected (fractions 1 through 4) library was 1,650 bp, with the longest being 4.4 kb. About 1/3 of the inserts from the size selected library were greater than 2 kb (Table 2, left side). Thus, a large enrichment for long cDNAs has been made by the size selection process. Surprisingly, about 1/3 of the inserts from the size selected library were also less than 1 kb, even though very little of the cDNA from fractions 1 through 4 appears to be this small (Fig. 3). The average size of inserts from this library also seems to be smaller that the apparent average size of the cDNA in fractions 1 through 4. A similar observation can be made for the average sizes of inserts from the total library. Two factors may contribute to this discrepancy. First, analysis of the insert sizes of random clones will lead to a number average size for the inserts, whereas the display of homogeneously labeled cDNA on a gel will lead to an apparent mass average size. The mass average is inherently larger than the number average, since a 2 kb cDNA will contain twice the radioactivity of a 1 kb cDNA. Second, longer cDNAs may be less clonable than shorter ones. In this case, construction of size selected libraries might be especially crucial in the expression cloning of cDNAs derived from long messages, and a low background vector such as the one we are describing would be especially useful in the construction.

Table 2. Size distribution of inserts before and after retroviral transduction. The number of clones (#) analysed which contained inserts in each size range and the percentage of clones (%) within each size range are shown. The source of clones before and after transduction is described in the text. *One of these inserts was longer than 3,000 bp and one was longer than 4,000 bp. size of insert

0-999 bp 1,000-1,999 bp 2,000 bp or more

(BEFORE) %

#

31 38 31

13 5 0

5 6 5*

(AFTER) % 72 28 0

3408 Nucleic Acids Research, Vol. 19, No. 12 Retroviral transduction of the size selected cDNA library Efficiency of retroviral transduction. The XZd vectors were originally designed to efficiently and stably transform a large range of cell types by retroviral transduction (8). For reasons which are not relevant to the present discussion, we attempted to transduce a derivative of the rat hepatoma cell line Fao (14) with the size selected A9 library. Since rat cells are relatively poorly infectable by these retroviruses, we chose a two step approach in which the library was first transduced, by transient expression using the Wgd5 producer (8), into the amphotrophic producer cell line PA317 (13). By selection with G418, a library of stable producers representing about 160,000 independent colonies of PA317s was thus established. These colonies were pooled and used as a source of high titer virus to transduce the Fao derivative cells by co-cultivation. In this second step transduction, about 225,000 G418 resistant colonies were obtained. cDNA length after retroviral transduction. Individual colonies of Fao cells were isolated, and the Zd inserts were rescued by WOP cell fusion (8) and plasmid DNA was digested with EcoRI and BamHI to excise rescued cDNA inserts. As shown in Table 2, these cDNA inserts were, on average, smaller than those of the original size selected library. Thus, it seems that the short cDNAs (

p lambda Zd39: a new type of cDNA expression vector for low background, high efficiency directional cloning.

We have developed a new type of bacteriophage lambda vector which provides a strong biological selection against non-recombinants that is independent ...
1MB Sizes 0 Downloads 0 Views