Isolation of genomic sequences flanking a retroviral insertion site using a novel PCR-based method

Gents. 161 (1995) 1955198 9 1995 Elsevier Science B.V. All rights reserved.

195

0378-I lI9~95~$09.50

GENE 08983

Isolation of genomic sequences flanking a retroviral insertion site using a novel PCR-based method (Mouse

genome; insertional

Dipa Natarajan

mutagenesis)

and Catherine

Received by R.W. Davies: I September

A. Boulter

1994; Revised/Accepted:

22 October114

November

1994: Received at publishers:

10 April 1995

SUMMARY

A novel PCR approach primers

and defined

the multiple

cloning

PCR amplification can be adapted

for the isolation

PCR conditions. site of pUC-based using primers

to enable

of genomic

The method plasmids,

sequences

that flank retroviral

is based on the directional isolation

of recombinant

of any genomic

sequences -

have mutagenesis

provides

a powerful

tool for

the isolation of new genes involved in mammalian opment (reviewed by Meisler, 1992). It is based integration disruption

of foreign DNA into the host genome causing of an endogenous gene, resulting in a mutant

phenotype.

The disrupted

using the introduced particularly transgenic

powerful mouse

gene can then be cloned

DNA as a molecular approach

technology,

the resulting mutation of the whole animal.

Any piece of exogenous mutagen.

Corrrspontlr~ncr

ro:

Dr.

of Cambridge.

Tel. (44.1223)

when

DNA

although C.A.

Boulter.

Downing

333-932; Fax (44-1223)

by

tag. This is a combined

with

since the consequences

can be studied

an insertional

University

develon the

within

of

the context

can potentially

act as

preference

genomic

DNA

into and

DNA

from pooled

colonies

The method

is simple and

most notably three

sites uses two standard

of digested

LTR sequences.

tagged by insertion

viruses,

INTRODUCTION

Insertional

plasmid

specific for the pUC vector and retroviral

rapid isolation

insertion

cloning

of foreign

MoMuLV

important

(Shinnick

advantages:

for integration

DNA.

et al., 1981),

firstly,

there

near or into transcribed

is a genes

( Mooslehner et al., 1990); secondly, they usually integrate as single copies, unlike the concatemers often associated with plasmid

DNA

integration;

thirdly,

the integration

event does not cause rearrangements or deletions at the site of insertion. However, isolation of the genomic sequences that flank the insertion site by the conventional cloning method of genomic library construction can be both

laborious

and

time-consuming.

In this paper

we

describe a simple and rapid approach which we have used to isolate genomic sequences adjacent to a retroviral integration

site.

vectors based on retroDepartment

Street, Cambridge

of

Genetics,

CB2 3EH. UK.

EXPEfRIMENTAL

AND DISCUSSION

333-992:

e-mail: [email protected] Abbreviations: Ap, ampicillin; bp, base pair(s); ES, embryonic stem: kb. kilobase or 1000 bp; LA, L-agar (see Sambrook et al.. 1989); LTR. long

terminal

repeat;

MCS,

multiple

cloning

site(s);

MoMuLV.

Moloney murine leukaemia virus; neo, gene encoding neomycin resistance: PCR. polymerase chain reaction: SOC (medium), see Sambrook et al. (1989). SSDI 0378.1

119( 95 )00265-O

(a) Mapping and cloning the proviral integration site In order to isolate genomic sequences flanking the MoMuLV-based retroviral vector N2 (Keller et al., 1985) in the ES cell line SR2-3 (Boulter et al., 1991), a map of the region was constructed, based on Southern analysis. This showed that, by digesting the SR2-3 genomic DNA

196 with Sal1 and BglII, a 3.9-kb fragment would be released which contained the neomycin-resistance-encoding gene (neo), the 5’ LTR of the retroviral vector and 1.2 kb of genomic sequence upstream from the insertion site. The Sal1 site is present at the 3’end of the neo gene and the BglII site is in the flanking DNA. Sal1 sites are less frequent in mouse genomic DNA than would be expected for a restriction enzyme that recognizes a 6-bp sequence, giving a significant enrichment for the desired fragment. The strategy for cloning this BglII-Sal1 genomic fragment is described in Fig. 1 and the legend thereto. The ligation was electroporated into bacteria and plated onto twelve Ap L-agar plates. After overnight incubation, 12 minipreps were made, one from each plate. PCR reactions were performed on each of the 12 plasmid samples generated, using two primers specific for the 5’ LTR (P2 and P3) in conjunction with the reverse primer of pUC1X (Pl), as shown in Fig. 2. PCR products of the expected size (1.3 kb with Pl and P2 and 1.6 kb with Pl and P3) were obtained with one of the samples, number 12. These were cloned and sequenced and were found to have MoMuLV LTR sequences present at one end (data not shown).

Digest with BgZII and SUB B,ijII

Sal1

S’LTR

neo

Ligate

i Transform into electrocompetent Plate onto Ap LA plates Pool ApRcolonies plate and prepare

bacteria

from each plasmid DNA

PCR using LTR and pUC 18 primers

Fig. 1. Strategy integration

for isolation

of genomic

sequences

site in the SR2-3 cell line. The positions

Pl, P2 and P3 are indicated

by arrowheads

flanking

and their sequences

given in the legend to Fig. 2. The BgfII + SalI-digested DNA was ligated loss of the BamHI then digested

to pUC18

with Sal1 +BamHI, vector.

The ligated

This was an enrichment

having a BamHI site would be linearized,

ized pUC18,

are

SR2-3 genomic resulting DNA

in was

with BamHI, a site known to be absent from the flanking

DNA and the N2 provirus. fragment

digested

site in the plasmid

the N2

of the PCR primers

and would therefore

transform

step; any genomic as would recircular-

bacteria

inefficiently.

The

ligated DNA was then electroporated into bacteria and plated on 12 L-agar (LA) plates with Ap selection, giving a total of 7.5 x lo4 resistant colonies

after over-night

incubation.

The bacterial

colonies

from each

(b) Confirmation of the proviral flanking sequence To confirm that the flanking sequence adjacent to the N2 provirus had indeed been cloned, two further primers (P4 and P5) were synthesized, based on the flanking sequence. These were used, together with a neo primer (P6), on genomic DNA from the SR2-3 cell line and on wild-type mouse DNA from the same strain (129). As shown in Fig. 3, PCR products of the expected size were obtained; the flanking primers, P4 and P5, gave a band of 0.45 kb in SR2-3 and normal DNA, whereas with primers P4 and P6 a band of the correct size, 2.0 kb, was obtained only in the SR2-3 DNA. Duplicate gels of these samples were blotted and hybridized with a neo probe and with a probe specific for the flanking sequence (Fig. 4). As expected, whilst the flanking probe hybridized to both fragments, the neo probe was specific for the 2.0-kb band (Fig. 4A,B). Moreover, when the 2.0-kb PCR product was isolated and digested with XbaI, which recognizes a site in the MoMuLV LTR, fragments of the correct size (0.7 and 1.3 kb) were generated, the smaller

plate were pooled in 10 ml of SOC medium, allowed to grow for 5 h in liquid culture at 37”C, and plasmid DNA isolated from each pool. Methods: 10 ug of genomic DNA, isolated from SR2-3 cells using a protocol

described

by Mullenbach

et al. (1989), was digested to comple-

tion overnight with 30 units each of BgBI + Sal1 under standard conditions. 1 pg of pUC18 was digested with BamHI+SalI and the vector fragment electroeluted from a 0.8% agarose gel. The ligations were performed over-night at 16°C with 300 ng of genomic DNA, 50 ng of vector and 0.8 units of T4 DNA ligase. The ligated DNA was digested for 3 h with BamHI, and half used for electroporation of MC1061. Electrocompetent bacteria (efficiency of 5 x 10’ transformants/ug DNA)

prepared according to Dower (1988), were electroporated using a BioRad Gene Pulser set at 200 uF, 25 f2 and 2.4 kV, using cuvettes with an electrode gap of 2 mm. After recovery in SOC medium for 1 h at 37°C the bacteria were plated on twelve 150mm diameter LA plates with 100 ug Ap/ml. A total of 7.5 x lo4 resistant colonies were obtained. Plasmid DNA was isolated from the pooled colonies from each plate according to Sambrook et al. (1989) and used for PCR with primers specific for the LTR and for pUC18, as described in the legend to Fig. 2.

197

M

A Pooled plasmid -

1

123456

DNA

h

12M

kb Primers Pl and P3

B -

-

I

kb

12M

P6

Primers Pl and P2

rl

*+

En

Fig. 3 PCR reactions

P2 P3 -4

cell line and

sequence

primers

PUC primers

neo

LTR

Fig. 2. PCR reactions

on pooled

specific for the pUCl8

plasmid

PUC

DNA samples

1 to 12, using

vector (Pl) and the LTR (P2 and P3 ).

( 1.6 kb). (B) PCR with Pl and P2, sample 12 giving a product

the expected

size (1.3 kb). A map of the 5’ flanking

vector cloned into pIJCl8 are indicated using

approx.

Methods: PCR reactions

10 ng of plasmid

2 units of Tcdq polymerase by the manufacturer per ml gelatin3

sequence

is shown below. The positions

by arrowheads.

DNA,

25 pmol

(Bioline, London,

containing

mM MgCI,.

from

with genomic

isogenic

of

and N2

of the primers were performed

of each

DNA isolated

129 mouse

liver. using

P4 and P5 and the wo primer

control,

plasmid

flanking

P6 as given in the

1, negative

DNA containing

from the

control,

the flanking

no DNA; sequence;

3

and 4. SR2-3 genomic DNA; 5 and 6, normal mouse DNA (129). Lanes 1. 2, 4 and 6, Primers P4 and P5; lanes 3 and 5. Primers P4 and P6.

Lanes: -. negative control, no plasmid DNA; M, I-kb DNA ladder. (A) PCR with PI and P3, sample 12 giving a product of the expected size

performed

legend to Fig. 2. Lanes: M, 1-kb ladder; 2, posttive

LTR

neo

m

PI *

I

I LTR

SR2-3

X

primer

Expected

sizes of PCR products:

2.0 kb with primers genome

0.45 kb wtth primers

P4 and P5, and

P4 and P6. A map of the N2 integration

of the SR2-3 cell line is outlined

is shown as a dark line. The positions

below. The flanking

of the flanking

for the PCR reactions

legend to Fig. 2. 100 ng of genomic

sequence

DNA probe and

neo probe (see Fig. 4) are shown as a black and hatched tively. Methods: Conditions

site in the

boxes, respec-

were as given in the

DNA and IO ng of plasmid

DNA

were used.

and

UK) in the buffer supplied

5 mM KCI/l

mM Tris pH 7;O.Ol mg

The PCR conditions

used were: 95’C for

5 min, followed by: 93 C for 30 s. 65’C for 50 s, 7O’C for 6 min. repeated

(c)

Conclusions (I) This method

provides

a rapid

and easy means

to

63 C for SO s. 7O’C for 6 min; 93’C for 30 s, 62’C for 50 s, 70-C for 6

isolate genomic sequences that flank retroviral insertion sites and it could be modified to clone any piece of foreign

min: 93 C for 30 s. 61 C for 50 s. 70 C for 6 min; 93 C for 30 s, 60‘C

DNA. Only a preliminary

3 times; 93 C for 30 s, 64’C for 50 s, 70‘ C for 6 min; 93-C for 30 s.

for 50 s. 70 C for 6 min. repeated 30 times. Primers used in the PCR reactions were as follows: PI (pUC18 reverse primer) 5’-ACACAGGAAACAGCTATGACCATG; GATCTG:

P?,

5’.GCAGTGGTGTCACATGCCTC TAACCTG:

P2,

S’CCATCTGTTCCTGACCTT-

5’CAAGAGGG’TTTATTGGATACACGGG: P5,

P4.

S’-GCTGTGATTGGGG-

P6,5’-GGCATCAGAGCAGCCGATTGTCTG.

of which hybridized with the flanking probe and the larger with the neo probe (Fig. 4C,D). This result demonstrated conclusively that the N2 flanking sequence had been cloned. This sequence was subsequently successfully library.

used

as a probe

to screen

a mouse

genomic

formed to obtain integration site.

Southern

blot needs to be per-

a simple map of the region flanking

the

(I) By using directional cloning, there is no need to size fractionate

which is very efficient, the genomic DNA. It

is possible

vector insertion

to clone one particular

site if

several exist, by the choice of suitable restriction enzymes. Enrichment for the correct insert is also possible for example, by using the relatively rare Sal1 site at the 3’ end of the neo gene or by digesting the ligation. (3) The method is generally applicable since the selection of suitable restriction enzyme sites in the flanking DNA is only limited by their presence in the MCS of the plasmid vector; given that there are many pUC-based

198 B

A kh

ACKNOWLEDGEMENTS

123456

123456

kb

We would like to thank the Lister Institute of Preventive Medicine and National Kidney Research Fund for supporting this work, and also Jim Murray for helpful comments on the manuscript.

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