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`Merck Ex. 1060, pg 1455
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`© Nature Publishing Group1979
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`Merck Ex. 1060, pg 1456
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`© Nature Publishing Group1979
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`Merck Ex. 1060, pg 1457
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`© Nature Publishing Group1979
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`Merck Ex. 1060, pg 1458
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`© Nature Publishing Group1979
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`Merck Ex. 1060, pg 1459
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`40
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`Nature Vol. 281 6 September 1979
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`tryptic peptides. A 1-ml
`Fig. 5 Fingerprints of
`aliquot of SVB"‘°‘«3-infected
`cell
`extract was
`immunoprecipitated as described in Fig. 4 legend,
`eluted in SDS sample buffer. mixed with Sug of
`unlabelled mouse globin and electrophoresed through
`a 20% polyacrylamide-SDS gel. A sample of authen-
`tic 35S—methionine-labelled mouse B“““-globin from
`induced Friend cells was electrophoresed in a separate
`track. The globin bands were identified by staining,
`excised, washed with 10% methanol, then digested in
`situ with trypsin—TPCK (Worthington)“. The super-
`natants were lyophilised, resuspended in 25 ul of
`H20 and analysed by two-dimensional ascending
`chromatography on 20x20 cm cellulose thin-layer
`plates (Brinkman). The first solvent was isoamyl
`alcohol/pyridine/H20 (9O:120:105)22. The second
`solvent was
`butanol/pyridine/ acetic
`acid/H10
`(32.5:2S:5:20) containing 7% (w/v) 2,5-diphenyl-
`oxazole“. The autoradiographs were exposed for 8 d
`at -70 "C. a, Globin from SVl9““"-3-infected cells; b,
`authentic mouse B"“‘-globin; c, mixture of globin
`from SVB"""-infected cells and authentic mouse 3"’
`globin (one-half the amounts used in ct and b).
`
`can be inserted into the late region of the recombinant if it is to
`be encapsidated into virions. This obstacle might be overcome
`by propagating the hybrid genome as an episome in persistently
`infected monkey cells’, thus overcoming the requirement for
`encapsidation. Furthermore, it is clear that, although we have
`
`concentrated on the globin system, the approach should also be
`applicable to a variety of other genes.
`We thank Drs K. Smith and S. Boyer for anti-B -globin
`antibody, B. Norman for preparing probes, and Ms C. Harvey
`for assisting in the preparation of the manuscript.
`
`Received 23 April; accepted 6 August 1979.
`
`.‘7‘5".‘*E"!°!"
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`
`
`Rabbit 3-globin mRNA production in
`mouse L cells transformed with
`cloned rabbit B-globin chromosomal DNA
`Ned Mantei, Werner Boll & Charles Weissmann
`Institut fiir Molekularbiologie I, Universitat Ziirich, 8093 Zfirich, Switzerland
`
`
`Mouse thymidine kinase-negative L cells were transformed
`with a cloned rabbit chromosomal B-globin gene linked to
`the cloned thymidine kinase gene of herpes simplex virus
`type 1. Most thymidine kinase—positive cell lines contained
`one or more copies of rabbit B-globin DNA and produced
`up to 2,000 copies of rabbit B-globin RNA per cell
`indistinguishable from its authentic counterpart. No mouse
`B—globin mRNA was detected.
`
`
`RECOMBINANT DNA technology has made it possible to iso-
`late, amplify and determine the primary structure of any
`eukaryotic DNA segment of interest. To explore the functional
`
`0028-0836/79/360040-07501.00
`
`significance of particular regions of a genome, it is desirable to
`generate localised modifications in the nucleic acid and then
`evaluate the resulting changes in the biological properties of the
`genome, in vitro or in viva (see ref. 1). The reintroduction of
`defined nucleic acid segments, in particular of cloned DNA, into
`eukaryotic cells has been accomplished by various methods.
`Mechanical microinjection of RNA or DNA into oocytes” or
`into somatic cells‘ delivers a large dose of nucleic acid to a
`relatively small number of identifiable cells. Biophysical and
`biochemical approaches use liposomes” or erythrocytes’
`loaded with RNA which are fused with the target cells, or direct
`treatment of target cells with nucleic acid in the presence of
`facilitating agents3’“. Efficient delivery of DNA to a large
`population of cells is attained by the use of DNA incorporated
`into viral particles”.
`
`© Macmillan Journals 1979
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`Merck Ex. 1060, pg 1460
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`Merck Ex. 1060, pg 1460