In situ transcription in cells and tissues

In situ transcription is provided by hybridizing cells or tissue with a primer, and extending the primer bound to any template mRNA or DNA with reverse transcriptase or DNA polymerase in the presence of labeled nucleotides, which allows for detection of cells containing the template. The resulting cDNA may be eluted and used in a polymerase chain reaction for isolation of the DNA, and/or the cDNA electrophoresed, which may provide for information concerning the sequence, or the like.

INTRODUCTION 
1. Technical Field 
The subject invention concerns in situ transcription for cDNA synthesis in 
fixed cells. 
2. Background of the Invention 
The rapid advances in molecular biology have opened up many avenues for 
investigation of a wide variety of physiological processes. In order to 
understand how various cells fulfill their functions, methods are being 
devised which allow for the detection of genes. One method has been the 
isolation of tissue, extraction of messenger RNA and the reverse 
transcription of the messenger RNA to produce cDNA. The cDNA may then be 
copied to provide double stranded(ds) cDNA which may be cloned. By 
traditional techniques, one often cannot identify the expression of a 
particular nucleic acid gene product in a subset of cells without a 
dilution effect resulting from tissue homogenization. Nor can they be used 
to identify individual cells containing a particular nucleic acid 
sequence. There is, therefore, substantial interest in being able to 
detect in tissue the distribution of expression of one or more products, 
as well as the ability to identify these products and obtain genes which 
may be used to express the products for further analysis and 
characterization. 
RELEVANT LITERATURE 
Reverse transcriptase can be used to produce a DNA sequence from RNA. 
Baltimore, Nature (1970) 26:1209. The polymerase chain reaction is 
described by Saiki et al., Science (1988) 239:487. See also, Eberwine, et 
al., Proc. Natl. Acad. Sci. USA (1987) 84:1449-1453. 
SUMMARY OF THE INVENTION 
In situ transcription is provided in which reverse transcription of mRNA is 
achieved in tissue sections or cells. Tissue sections or cells are 
obtained, the mRNA in the cells hybridized with an appropriate primer, and 
then contacted with reverse transcriptase, employing labeled nucleotides, 
particularly radioactive labels. The resultant signal intensities may be 
related to the level of mRNA in specific portions and/or cells of the 
section, while the transcripts may be eluted from tissue sections for a 
variety of uses. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
In accordance with the subject invention, in situ transcription is 
performed with tissue sections. The tissue sections are contacted with a 
nucleotide primer under hybridizing conditions, followed by contacting 
with reverse transcriptase under primer-extension conditions. By employing 
a label, particularly radioactive nucleotides, the primer extension can be 
followed in the tissue section, demonstrating those cells and sections in 
the tissue in which the mRNA's are present and providing a qualitative 
estimation of the mRNA present. If desired, the transcripts may be 
isolated by elution and used in cloning, for expression, or the like. 
The tissue sections may be obtained by any convenient manner: cryostat 
sections, microtome slices or the like may be prepared. The source of 
tissue may be any solid organ, such as brain, spleen, bone, heart, 
vascular, lung, kidney, liver, pituitary, endocrine glands, lymph node, 
dispersed primary cells, tumor cells, or the like. The tissue may be 
pretreated in conventional ways prior to sectioning to provide the tissue 
section sample. Usually a tissue section will be at least about 1 .mu.m 
thick, and not more than about 50 .mu.m usually not more than about 25 
.mu.m, and preferably from about 5 to 15 .mu.m thick. The section may then 
be fixed by convenient techniques which do not interfere with the 
subsequent processing. Conveniently, aldehyde (e.g. dilute aqueous 
formaldehyde or acetaldehyde) fixing or other conventional technique may 
be employed. The particular manner in which the tissue is fixed is not 
critical to this invention, so long as the procedure does not destroy the 
mRNA, nor interfere with the subsequent steps of the process. 
After the tissue is fixed, it may then be hybridized with the appropriate 
primer. Where it is desired to obtain polyadenylated mRNA, a poly-T primer 
may be employed. For the most part one will be interested in one or a few 
mRNA's and in this situation a specific oligonucleotide or a mixture of 
oligonucleotides coding for a single oligopeptide may be employed. 
Depending upon the specificity desired, various levels of stringency may 
be employed in the hybridization. The stringency may be as a result of 
elevated temperatures, salt concentrations, organic solvents, or the like. 
Conveniently, the hybridization medium may have from about 10 to 75% of a 
polar organic solvent, e.g., formamide. Salt concentrations will generally 
vary from about 0.4M to 1M. The time for the reaction will generally vary 
from about 1 to 48 hours, more usually from about 6 to 24 hours. The 
temperature will generally vary from about 20.degree. to 60.degree., more 
usually from about 20.degree. to 40.degree. C. The amount of primer will 
vary widely, generally from about 0.1 ng, more usually from about 0.1 to 
25 ng per tissue section of 30 .mu.L volume of tissue of the hybridizing 
medium. The volume employed will vary depending on the size of the tissue 
section, usually within the range of about 5 to 200 .mu.l, and more 
usually from about 5 to about 100 .mu.l. 
The primer will generally be at least about 12 nucleotides (nt), and 
usually not more than about 200 nt, more usually not more than about 100 
nt and preferably not more than about 50 nt. The primer may be labeled or 
unlabeled, conveniently unlabeled or are radioactive, or the like. If 
labeled, various labels may be employed which have a specific receptor 
which may be detected or are radioactive or the like. For example, the 
probe may be labeled with biotin, which may subsequently be bound with 
avidin which may be labeled with fluorescers, enzymes, or the like. 
Fluorescers of interest may include phycobiliproteins, fluorescein, or the 
like. Enzymes may include horseradish peroxidase, phosphatase, or the 
like. Where the enzyme is likely to be encountered in the tissue cells, 
prior to hybridization, the protein of the tissue may be denatured. 
Denaturation can be achieved with heat, specific denaturants, or the like. 
In some instances the manner in which the tissue was fixed will suffice 
for denaturation. 
Once the hybridization has been carried out, the sections may be washed one 
or more times, where the washes may provide for the desired stringency. 
Generally, the washes will have a salt concentration which may be the same 
as or less than the salt concentration employed during the hybridization 
and may have a temperature in the range employed for the hybridization, 
preferably from about 25.degree. to 45.degree. C. Desirably, the washes 
may be monitored to detect the continued presence of the primer in the 
wash. 
Reverse transcription may then be carried out under conventional conditions 
for reverse transcriptase. Reverse transcriptase is commercially available 
and the supplier provides the preferred conditions. Conveniently, the 
reverse transcription may be carried out at an elevated temperature, in 
the range of about 25.degree. to 40.degree. C. for a time in the range 
from 0.1 to 6 h, more usually from about 0.5 to 3 h. 
Instead of reverse transcriptase, a DNA polymerase may be used which is 
functional in the cell. Either an intact DNA polymerase or a functional 
fragment thereof, e.g. the Klenow fragment, may be employed. Various DNA 
polymerases are commercially available and may be used in accordance with 
conventional conditions. While throughout this specification, cDNA will be 
referred to as copied from mRNA, it is to be understood that DNA may also 
be copied to provide dsDNA, with the complementary strand being the 
equivalent of the cDNA reverse transcribed strand. 
Included in the reaction mixture will be the necessary deoxynucleotide 
triphosphates where one or more of the nucleotides may be labeled, 
particularly with a radioactive label, such as .sup.35 S, .sup.32 P, 
.sup.3 H, or the like. Alternatively, nucleotides may be obtained which 
are labeled with biotin, where these nucleotides will become incorporated 
in the extended chain. The biotin may then be used for binding to avidin, 
which is labeled with an appropriate, label capable of providing for 
detection, as described previously. After completion of the reverse 
transcription, the sections are thoroughly washed to remove any of the 
labeled nucleotide monomer. Washes will generally be dilute salt solutions 
less than about 0.25M, at ambient or elevated temperatures, usually not 
exceeding about 60.degree. C. 
Depending upon the nature of the label, the sections may be treated 
differently. Where a radioactive label is employed, the section may be 
dehydrated and apposed to appropriate x-ray film. With other labels, the 
tissue section may be soaked with the appropriately labeled receptor for a 
sufficient time for specific binding to occur, followed by thorough 
washing, so as to ensure the complete removal of any non-specifically 
bound labeled receptor. The washes may be selected so as to selectively 
remove the labeled receptor without removing the receptor bound to the 
nucleic acid. 
To characterize the transcripts, various procedures may be employed. The 
transcripts may be eluted from the sections and separated on a denaturing 
gel. The cDNA transcripts are found to form a complex banding pattern in 
at least some instances. Desirably, the cDNA may be modified to provide a 
template which may then be used in a polymerase chain reaction to greatly 
expand the amount of cDNA produced. The resulting cDNA may be used for 
further cloning, expression, or the like. The subject methodology can 
obviate the need to make cDNA libraries of high complexity for specific 
cDNA isolation. The subject method provides for the direct transcription 
of messenger RNA in tissue in accordance with the selection of the primer. 
Thus, only one or a few different sequences will be obtained, which may 
vary as to the degree of their extension. 
The banding pattern in the electrophoresis may also afford an opportunity 
for differentiation of various messenger RNA's. Patterns may be associated 
with unique structural aspects of the template, being, for example, 
related to viral subtypes, alternately spliced products, secondary 
structure, association of binding proteins, such as ribosomes with the 
mRNA, or the like. Thus, the subject method may be used in a variety of 
ways to evaluate a physiological status of cells in tissue sections to 
identify the pattern of expression of one or more proteins in the cells 
and tissue sections, to provide for the isolation of genes encoding for a 
protein encompassing a particular oligopeptide, or the like. 
The following examples are all by way of illustration, not by way of 
imitation.

EXPERIMENTAL 
Cryostat sections (11 .mu.m thick) were prepared from fresh frozen rat 
pituitaries. Sections were fixed for 5 min in 3 percent neutral buffered 
paraformaldehyde. In situ hybridization was performed for 12 to 16 hours 
at room temperature in a mix consisting of 4.times.SSC/50% formamide, to 
which was added one ng unlabeled oligonucleotide in 25 .mu.l. Sections 
were washed with two changes in 2.times.SSC for 30 min at room 
temperature, followed by washes in 0.5.times.SSC at 40.degree. C. for two 
hours with one change. Reverse transcription was performed at 30.degree. 
C. for one hour in a reaction mix consisting of 15 mM tris-HCl, pH 8.3, 6 
mM MgCl.sub.2, 40 mM KCl, 7.5 mM dithiothreitol, 250 mM dATP, dTTP and 
dGTP, 300 .mu.Ci/ml .alpha.--[.sup.35 S]dCTP (1,000 Ci/mmol, Amersham), 
0.12 units ribonuclease inhibitor (RNasin)/.mu.l (BRL, Bethesda, Md.), and 
600 units/ml avian myoblastosis virus reverse transcriptase (Seikagaku, 
St. Petersburg, Fla.). Sections were then washed, with two changes of 
2.times.SSC for 30 min each at room temperature followed by washes in 
0.5.times.SSC at 40.degree. C. for 6 hours with two changes. Sections were 
then dehydrated and apposed to Kodak XAR x-ray film for 10 min prior to 
development. 
Drug treatments: Rats received daily intraperitoneal injections of 
bromocriptine (Sandoz) (3 mg/kg body weight), haloperidol (McNeil) or 
vehicle for four days and were sacrificed 24 hours after the last 
injection. 
For localization by emulsion autoradiography, the above procedure was 
followed, except that 60 .mu.Ci/ml .alpha.--[.sup.3 H]dCTP (50 Ci/mmol, 
Amersham) replaced the .sup.35 S-labeled nucleotide in the reverse 
transcriptase reaction. The section was dipped in Kodak NTB2 nuclear track 
emulsion diluted 1:1 with water. Following a 15-hour exposure, the 
autoradiogram was developed in Kodak D19 for 2 min at 17.degree. C. and 
fixed in Kodak fixer for 5 min at 17.degree. C. Counterstaining was 
performed with hematoxylin and eosin. 
For electrophoresis of the transcripts, the following procedure was 
employed: The procedure was performed as described above, except that 300 
.mu.Ci/ml .alpha.--[.sup.32 P]dCTP (410 Ci/mmol, Amersham) replaced 
.alpha.--[.sup.35 S]dCTP in the reverse transcriptase reaction. For gel 
electrophoresis, transcripts were denatured by incubating sections in (50 
.mu.l per section) 4M guanidine-HCl/1M .beta.-mercaptoethanol for 1 hour 
at 23.degree. . This was followed by protein extraction with 0.5 vol 
phenol/0.5 vol chloroform, with subsequent ethanol precipitation (10 mg/ml 
glycogen, 2 vol ethanol, 30 min in dry ice) prior to electrophoresis. 
The proopiomelanocortin (POMC) mRNA was used to evaluate the subject 
method. The POMC gene gives rise to a family of biologically active 
peptides including adrenocorticotropin, .beta.-endorphin and 
.alpha.-melanocyte-stimulating hormone. An oligonucleotide 36-nt in 
length, complementary to the sequence encoding amino acids 100 to 111 of 
rat POMC was used as the primer. After in situ hybridization to rat 
pituitary tissue as described above, a strong signal was observed in the 
intermediate lobe consistent with the known localization of POMC mRNA. 
When the primer was omitted from the hybridization mix, the signal was not 
observed. Omission or heat-inactivation of the reverse transcriptase also 
eliminated the signal. As a negative control, reverse transcription was 
performed after a hybridization step with a 36 nt probe complementary to 
tyrosine hydroxylase mRNA, which has not been detected in the rat 
pituitary. No signal above background was produced. As a positive control, 
hybridization was performed with a polythymidine (poly-T oligomer) 36 nt 
in length. Intense signals in both the anterior and intermediate lobes 
were observed, consistent with the abundance and expected distribution of 
poly(A.sup.+) mRNA. 
To further confirm the specificity of the POMC signal, in situ 
transcription was performed in pituitary sections from animals that had 
been treated with the dopamine antagonist haloperidol and the dopamine 
agonist bromocryptine. Various samples were employed and electrophoresed: 
(A) 5% polyacrylamide/7M urea gel, 3000 cpm loaded per lane: transcripts 
resulting from priming with 
(a) no oligonucleotide; 
(b) heterologous 36 nt oligonucleotide; 
(c) POMC oligonucleotide. 
(B) 5% polyacrylamide/7M urea gel: comparison of 
(d) POMC-primed cDNA transcribed in the presence of .alpha.-[.sup.32 P]dCTP 
(3000 cpm loaded); and 
(e) transcripts primed by a 5'-end, .sup.32 P-labeled POMC oligonucleotide 
(specific activity, 2.times.10.sup.8 cpm/.mu.g), followed by reverse 
transcription in the presence of 250 .mu.M unlabeled deoxynucleotides (900 
cpm loaded). 
(C) 6% polyacrylamide/7M urea gel: comparison of 
(f) POMC-primed transcripts after elution from tissue; and 
(g) POMC-primed transcripts after elution from tissue and hybridization to 
POMC cDNA that had been immobilized on nitrocellulose filters; after 
hybridization in 30% formamide, 5.times.SSC, 100 .mu.g/ml salmon sperm DNA 
and 5.times.Denhardts, the filters were washed in 2.times.SSC, 0.1% SDS 
for 30 min at 37.degree. C. followed by 2 washes in 0.2.times.SSC, 0.1 SDS 
for 30 min at 42.degree. C., and the bound transcripts were eluted by 
incubating the filter in a solution containing 0.1% SDS, 0.5 mM EDTA at 
65.degree. C. for 30 min, followed by ethanol precipitation; and 
(h) transcripts that did not hybridize to the POMC-cDNA-bound filter. 
The intensities of the intermediate-lobe signals varied in a manner 
consistent with the known dopaminergic regulation of POMC mRNA. The 
neuroleptic haloperidol, which elevates POMC mRNA levels, increased the 
intermediate lobe signal relative to control. Conversely, the dopamine 
agonist bromocryptine greatly reduced the signal, consistent with the 
effect of this drug on intermediate lobe POMC mRNA levels. 
High intensities of the intermediate-lobe POMC signal were observed. 
Exposure times of 10 min were required for film autoradiograph with 
.sup.35 S-labeled transcripts, in contrast with the 3-hour exposures 
required for in situ hybridization with 3'-end, .sup.35 S-labeled 
oligonucleotide probes. When the subject reaction was performed in the 
presence of tritiated deoxycytidine, an emulsion autoradiographic exposure 
of only 15 hours was sufficient for the cellular localization of silver 
grains in the intermediate lobe. The increase in signal intensity relative 
to standard protocols likely resulted from the incorporation into the 
transcripts of many radio-labeled deoxynucleotides (approximately 25% of 
incorporated deoxynucleotides) per mRNA molecule. 
A primer-independent background signal was observed, which did not require 
a hybridization step prior to the enzyme reaction, but did require reverse 
transcriptase. This suggests that endogenous sites occurred in the tissue 
section, serving as primer-template complexes for the initiation of 
reverse-transcriptase activity. 
To further characterize the transcripts produced in the POMC reaction, 
radiolabeled POMC oligonucleotide-primed transcripts were eluted from 
pituitary sections and separated on a denaturing gel. The cDNA transcripts 
from POMC primer formed a complex banding pattern, while transcripts 
produced after hybridization with the heterologous tyrosine hydroxylase 
oligonucleotide produced a smear of radioactivity, similar to that of the 
unprimed sample. 
To determine whether the bands represented extensions of the 
oligonucleotide primer, hybridization with a 5'-end .sup.32 P-labeled POMC 
oligonucleotide was performed, followed by reverse transcription in the 
presence of high concentrations of unlabeled deoxynucleotides. The 
resulting transcripts produced a similar autoradiographic pattern of 
bands, thus demonstrating that they resulted from extension of the 
oligonucleotide primer. 
To determine whether the POMC oligonucleotide-primed transcripts were cDNA 
copies of POMC mRNA, the denatured transcripts were hybridized to 
single-stranded POMC cDNA that had been immobilized on nitrocellulose 
filters. After high-stringency washes, the hybridized transcripts were 
eluted from the filter and electrophoresed on a polyacrylamide-urea gel. 
The banding pattern prior to hybridization was similar to that observed 
for transcripts that had hybridized to POMC cDNA, whereas the 
non-hybridizing transcripts did not produce bands on electrophoresis. This 
identified the bands as sequences complementary to POMC cDNA. 
It was further shown that the specific cDNA's produced by the in situ 
transcription reaction may be denatured and eluted from tissue sections 
for cloning. It was possible to clone POMC cDNA from 3 11 .mu.-thick 
pituitary sections using the following method: After eluting from the 3 
pituitary sections, the second-strand cDNA was synthesized by self-priming 
(Efstratiatis et al., Cell (1976) 7:279). This was followed by 
blunt-ending of the double-stranded cDNA with T.sub.4 DNA polymerase and 
subsequent restriction of the cDNA with HaeIII. (Seeburg et al., Nature, 
220:486). These fragments were then cloned into SmaI-linearized pSP64, 
whereupon transformation into DH5a cells, followed by colony lifts and 
hybridization with a POMC gene-fragment probe (Roberts et al. in Recent 
Progress in Hormone Research (Academic Press, NY, 1982), p. 227) revealed 
that 40% of the insert-containing transformants contained POMC cDNA. The 
cDNA was approximately 70 nt in length and resulted from cloning of the 
HaeIII fragment closest to the primer site. (Drouin and Goodman, Nature 
(1980) 288:610). 
Since these sections contained roughly 4.5 ng of poly(A.sup.+)RNA, cloning 
of specific cDNA's by in situ transcription may be achieved with a smaller 
amount of tissue than is required by other methods, which usually require 
greater than about 1 .mu.g of poly(A.sup.+) mRNA. 
The subject technique may be used for cloning human cDNA's from limited 
quantities of tissues obtained pre- or post-mortem to determine the 
precise anatomical localization of mRNA's and to demonstrate the 
specificity of transcription in tissues having a complex population of 
mRNA's, such as the brain. In addition, the subject technique may be 
combined with the polymerase chain reaction to avoid making highly complex 
cDNA libraries, to provide for convenient amounts of copies of rare mRNA, 
or the like. 
It is evident from the above results that the subject invention provides 
for a highly efficient manner for obtaining a large amount of information 
about transcription products from cells in tissue. The method provides for 
anatomical localization of the cells involved in transcription. In 
addition, the cDNA's produced may be eluted and used in a variety of ways. 
The eluted cDNA's may be electrophoresed, whereby a unique pattern of 
bands may be obtained. The band pattern may provide for differentiation of 
viral subtypes or alternate splice-products of a single gene, information 
concerning the secondary structure of the template or association of 
ribosomes or other proteins with the mRNA, or the like. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity of understanding, it 
will be obvious that certain changes and modifications may be practiced 
within the scope of the appended claims.