Correction to: Comparative analysis of confocal microscopy on fresh breast core needle biopsies and conventional histology.

Following publication of the original article [1], the authors reported an added data on Table 1 in their paper. The original article [1] has been updated.

Comparative analysis of confocal microscopy on fresh breast core needle biopsies and conventional histology.

BACKGROUND: Evaluation of core needle biopsies (CNB) is a standard procedure for the diagnosis of breast cancer. However, tissue processing and image preparation is a time- consuming procedure and instant on-site availability of high-quality images could substantially improve the efficacy of the diagnostic procedure. Conventional microscopic methods, such as frozen section analysis (FSA) for detection of malignant cells still have clear disadvantages. In the present study, we tested a confocal microscopy scanner on fresh tissue from CNB with intention to develop an alternative device to FSA in clinical practice. PATIENTS AND METHODS: In 24 patients with suspicious breast lesions standard of care image-guided biopsies were performed. Confocal images have been obtained using the Histolog™ Scanner and evaluated by two independent pathologists. Hematoxylin-Eosin (H&E) histological sections of the biopsies were routinely processed in a blinded fashion with respect to the confocal images. RESULTS: In total 42 confocal images were generated from 24 biopsy specimens, and available for analysis within a few minutes of taking the biopsy. This resulted in 2 × 42 = 84 pathologic evaluations. In four cases, a pathologic diagnosis was not possible with confocal microscopy. An exact correlation based on the B-classification was reached in 41 out of 80 examinations and in another 35 cases in a broader sense of correspondence definition (i.e. malignant vs. benign). CONCLUSIONS: As a reliable on-site method, the Histolog™ Scanner provides a visualization of cellular details equivalent to the H&E standards, permitting rapid and accurate diagnosis of malignant and benign breast lesions. Furthermore, this device offers great potential for immediate margin analysis of specimen in breast conserving therapy.

Disease-related surgery in patients with distant metastatic breast cancer.

INTRODUCTION: This study evaluates the frequency of and indications for disease-related surgical procedures in the palliative breast cancer (BC) situation. PATIENTS & METHODS: Based on a cohort of women who were treated for newly diagnosed BC during a 20-year period (1990-2009), we analyzed 340 patients who developed distant metastatic disease (DMD) until 2011 and died (i.e. still ongoing palliative disease courses were not included). RESULTS: One hundred and twenty-seven surgical procedures were performed in 100 patients (29.4% of all patients with metastatic disease). The most common site for surgery was breast (n = 60, 47.2%). The primary tumor was removed at first diagnosis of DMD in 43 patients (33.9%); sixteen operations (12.6%) were performed for local recurrence. In 37 patients, 50 surgical procedures (39.4%) were necessary to stabilize osseous structures due to metastases. Procedures were rarely performed on other common metastatic sites: lung: n = 1 (0.8%); liver: n = 1 (0.8%), brain: n = 4 (3.1%). When excluding surgery for primary breast tumors at initial diagnosis of DMD from analysis, 34 of 84 surgeries (40.4%) were performed in the first third of survival follow-up (i.e. period of metastatic disease survival); operations in the last two-thirds each totaled 29.8% (n = 25). The median survival after surgery was 16 months (range: 0.5-89 months). CONCLUSIONS: In a cohort of BC patients who had primary or developed secondary DMD, nearly one third of the patients received disease-related surgical procedures during their palliative disease course. This high rate of operations shows that surgery has a clearly established role in the palliative therapy concept.

Content comparison of health status measures for obesity based on the international classification of functioning, disability and health.

OBJECTIVE: To compare the content covered by twelve obesity-specific health status measures using the International Classification of Functioning, Disability and Health (ICF). DESIGN: Obesity-specific health status measures were identified and then linked to the ICF separately by two trained health professionals according to standardized guidelines. The degree of agreement between health professionals was calculated by means of the kappa (kappa) statistic. Bootstrapped confidence intervals (CI) were calculated. The obesity-specific health-status measures were compared on the component and category level of the ICF. MEASUREMENTS: welve condition-specific health-status measures were identified and included in this study, namely the obesity-related problem scale, the obesity eating problems scale, the obesity-related coping and obesity-related distress questionnaire, the impact of weight on quality of life questionnaire (short version), the health-related quality of life questionnaire, the obesity adjustment survey (short form), the short specific quality of life scale, the obesity-related well-being questionnaire, the bariatric analysis and reporting outcome system, the bariatric quality of life index, the obesity and weight loss quality of life questionnaire and the weight-related symptom measure. RESULTS: In the 280 items of the eight measures, a total of 413 concepts were identified and linked to the 87 different ICF categories. The measures varied strongly in the number of concepts contained and the number of ICF categories used to map these concepts. Items on body functions varied form 12% in the obesity-related problem scale to 95% in the weight-related symptom measure. The estimated kappa coefficients ranged between 0.79 (CI: 0.72, 0.86) at the component ICFs level and 0.97 (CI: 0.93, 1.0) at the third ICF's level. CONCLUSION: The ICF proved highly useful for the content comparison of obesity-specific health-status measures. The results may provide clinicians and researchers with new insights when selecting health-status measures for clinical studies in obesity.

Up to 21-day banked red blood cells collected by apheresis and stored for 14 days after automated wash at different times of storage.

BACKGROUND AND OBJECTIVES: A closed-system technology (ACP-215, Haemonetics, Braintree, MA) enables automated washing and extended storage of frozen red blood cells (RBC). This technology was applied to wash banked RBC for removal of undesirable protein and metabolites before transfusion. We studied protein and metabolite depletion as well as RBC metabolism and viability up to 14 days postwash with regard to various pre-storage times. MATERIALS AND METHODS: Thirty RBC units were collected by means of apheresis and subdivided into three arms based on prewash storage time period (6 days/group 1, 14 days/group 2, 21 days/group 3). Wash efficacy (protein depletion, IgA), RBC metabolism (pH, lactate, potassium, haemolysis) and cell viability (ATP) were analysed immediately and 14 days after washing. RESULTS: Total protein and IgA postwash were lowered by automated wash in all groups and uniformly met EC guidelines. Potassium (mmol/l) was below 1.2 mmol/l postwash and significantly below prewash values in all groups, even after 14 days of storage (prewash vs. postwash; P < 0.05). RBCs washed after 14 and 21 days, respectively, showed significantly lower pH values and lower ATP content than RBCs washed after only 6 days of storage. Haemolysis rate remained significantly below 0.8%, the maximum level recommended by the EC guidelines, immediately and 14 days after washing in all units. CONCLUSION: Our data confirm that RBC units banked up to 21 days can be effectively protein- and potassium-depleted with the ACP-215 independent from prewash storage time. With respect to high ATP levels and pH, postwash storage of 2 weeks should be limited to units not older than 7 days before wash. This new washing technology ensures better standardization in washed RBC and provides blood centres with a logistical alternative to 24-h washed RBC products.

Linking osteoarthritis-specific health-status measures to the International Classification of Functioning, Disability, and Health (ICF).

OBJECTIVES: The objective of this study was to link the Western Ontario and McMaster Universities (WOMAC) and Lequesne-Algofunctional indices to the ICF on the basis of linking rules developed specifically to accomplish this aim. The linking process enables the understanding of the relationship between health-status measures and the ICF. METHODS: Since the fifth World Health Organisation/International Liege Against Rheumatism (WHO/ILAR) Task Force and the Outcome Measures in Rheumatology Clinical Trials (OMERACT) group recommend the use of WOMAC and the Lequesne-Algofunctional indices in patients with osteoarthritis of the hip and knee in clinical trials, these two health-status measures have been used in this study. Both health-status measures were linked to the ICF separately by two trained health professionals. Consensus between health professionals was used to decide which ICF category should be linked to each item/concept of the two questionnaires. To resolve disagreements between the two health professionals, a third person trained in the linking rules was consulted. RESULTS: Except for the concept of 'morning stiffness', both health professionals agreed on the ICF category chosen to link all the items/concepts of both questionnaires. Altogether, 29 different ICF categories have been linked. Five ICF categories belong to the ICF component 'body functions', 23 categories to the component 'activities and participation', and one category to 'environmental factors'. Both questionnaires have 10 ICF categories in common. CONCLUSIONS: The results of the linking process reflect both the structure of the two questionnaires studied and the relationship between them, showing that the ICF classification can become the cardinal reference for existing health-status measures.

Mucin 7 and cytokeratin 20 as new diagnostic urinary markers for bladder tumor.

PURPOSE: We determine the sensitivity and specificity of cytokeratin 20 (CK-20) and mucin 7 (MUC7) gene expression in voided urine samples taken from patients with bladder tumor and from control groups to investigate putative, noninvasive urinary markers for bladder tumor detection and monitoring. MATERIALS AND METHODS: Voided urine samples were collected from 50 patients with histologically proven bladder neoplasms (pTaN0M0G1-3 in 19 and pTisN0M0G3-pT4pN1M1G3 in 31), 20 patients with urolithiasis, 20 patients with urinary tract infection, 20 patients with other urological neoplasms and 20 healthy volunteers. Total RNA was extracted from exfoliated cells collected from 200 ml. voided urine. All RNA samples were investigated by a specific CK-20 and MUC7 nested reverse transcriptase polymerase chain reaction. RESULTS: The overall sensitivity of CK-20 gene expression in voided urine samples for the detection of bladder neoplasms was 78%. In contrast, voided urine samples from control patients and healthy volunteers showed a high rate of false-positive CK-20 detection resulting in a low specificity of 36%. The overall sensitivity of the MUC7 test for all bladder tumor cases was 66%. The sensitivity for papillary urothelial neoplasms (pTaN0M0G1-3) was 42% whereas analysis of the carcinoma in situ and invasive bladder cancer group (pTisN0M0G3-pT4pN1M1G3) yielded a sensitivity of 81%. The overall specificity of the MUC7 nested reverse transcriptase polymerase chain reaction method in the control groups was 80%. CONCLUSIONS: A high positive CK-20 detection rate was found not only in voided urine samples from patients with bladder tumor, but also in urine specimens from control groups. Therefore, CK-20 is not a reliable urinary tumor marker for bladder neoplasms. In contrast to CK-20, analysis of MUC7 demonstrated a high sensitivity and high specificity for carcinoma in situ and invasive bladder cancer, thus fulfilling the criteria of a urinary tumor marker.

A new tumour marker assay for ovarian cancer on the OPUS immunoassay system.

The human tumour associated cancer antigen CA 125 is a glycoprotein with high molecular weight. The determination of this antigen has been proven to be useful in the monitoring of patients with ovarian cancer. OPUS OV, the tumour marker assay for the ovarian cancer antigen CA 125 is an ELISA that was developed for the family of fully automated random-access analyzers, OPUS, OPUS Plus and OPUS I Magnum. The assay uses a double monoclonal sandwich format (antibodies B27.1 and B43.13, Biomira/Canada). The first antibody is immobilized on the solid phase of the OPUS modules. Sample is automatically added and incubated for 5 minutes. The addition of a solution of the second antibody conjugated to the enzyme alkaline phosphatase leads to the formation of a sandwich complex within 5 minutes. The last step, the addition of the fluorogenic substrate 4-methylumbelliferyl phosphate, serves both as washing procedure and for the development of the fluorescence signal (kinetic measurement). OPUS OV has an assay range from 0-1000 kU/L with a detection limit of 5 kU/L. Within run cv's are 6-8%. A good correlation was found to commercially available kits for the determination of CA 125. We conclude that this new OPUS OV assay is a valid alternative for use in the routine determination of the cancer associated antigen CA 125 and allows more reliable determinations in terms of random access, speed, and ease of operation.

Properties of two insect cell lines useful for the baculovirus expression system in serum-free culture.

The properties of Sf9 and Tn5 insect cells were analyzed comparatively under serum-free culture conditions. Sf9 cells in SF900II medium apparently utilized sucrose as a primary nutrient both before and after virus infection, yielding small amounts of lactate and ammonia. Tn5 cells in Excell 401 medium consumed all the nutrients examined, including sucrose. The productivity of a recombinant glycoprotein, OSF-2, by Tn5 cells, was moderate in both monolayer and spinner cultures, but the ability to secrete it was compromised in the former case. Relative to the Tn5 cultures, Sf9 produced 30-fold more OSF-2 in either culture mode. (c) 1996 John Wiley & Sons, Inc.

Expression and characterization of murine osteoblast-specific factor 2 (OSF-2) in a baculovirus expression system.

Osteoblast-specific factor 2 (OSF-2) is a approximately 90-kDa protein selectively expressed in bone. OSF-2 cDNA was recently isolated from mouse and human cDNA libraries and shows limited sequence homology with fasciclin I, a cell adhesion protein expressed in insect nerve cells. Here we describe the expression of recombinant murine OSF-2 (rmOSF-2) in a baculovirus/insect cell system. Western blotting analysis employing polyclonal antiserum raised against a C-terminal synthetic OSF-2 peptide detected a protein of approximately 90-kDa as early as 2 days after infection of Sf9 cells with the recombinant virus. Tunicamycin treatment of infected cells resulted in a mobility shift of OSF-2 (approximately 90-kDa band) on Western blots. N-Glycanase digestion resulted in the same mobility shift of OSF-2, indicating that rmOSF-2 expressed in insect cells is N-glycosylated. However, OSF-2 was insensitive to endoglycosidase H digestion while a major fraction of this protein had affinity for concanavalin A. Finally, it was demonstrated that rmOSF-2 was able to bind to heparin. This finding suggests that OSF-2 might be associated with the bone extracellular matrix after secretion by osteoblasts and participate in cell adhesion and/or cell communication. The establishment of the baculovirus expression system with a high productivity of recombinant OSF-2 (around 40 micrograms/ml at maximum) and its heparin binding properties should allow us to obtain large amounts of rmOSF-2.

Sequences of two porcine glutamic acid decarboxylases (65- and 67-kDa GAD).

The cDNAs encoding the porcine 65- and 67-kDa glutamic acid decarboxylases (GAD65 and GAD67, respectively) were cloned by the PCR method. The 2246-nucleotide (nt) GAD65 cDNA contained an open reading frame (ORF) coding for a protein of 585 amino acids (aa), and the 3262-nt GAD67 cDNA contained an ORF coding for a protein of 594 aa. A remarkable conservation was shown when the deduced aa sequences of porcine GAD65 and GAD67 were compared with those of other mammalian species (human, cat and rat). Porcine GAD65 is 96% identical to human and rat GAD65, and porcine GAD67 is more than 95% identical to human, cat and rat GAD67 at the aa level.

Survival of genetically-engineered and wild-type strains of the yeast Saccharomyces cerevisiae under simulated environmental conditions: a contribution on risk assessment.

A genetically-engineered strain of Saccharomyces cerevisiae employed for the industrial production of the human coagulation Factor XIIIa (rhFXIIIa) was used for a survival study under simulated environmental conditions. The homologous strain devoid of the recombinant plasmid and the homologous strain bearing the 2 microns-based vector plasmid without the rhFXIIIa-encoding DNA insert were compared. The strains were introduced into natural soil/water suspension, into soil/medium suspension and into waste water. After intervals, samples of cell suspensions were taken and viable cell numbers were determined by plating on antibiotic-containing medium. In addition, a non-radioactive technique involving enhanced chemiluminescence was employed to detect plasmid-bearing yeast cells. The rhFXIIIa expression plasmid showed a high stability during the simulated environmental condition. No differences in survival rates, however, could be detected for the plasmid-bearing and plasmid-less strains under the three conditions tested, suggesting that the presence of plasmid does not confer selective advantages on the survival of the yeast cells. It is concluded that, even after accidental release of the engineered yeast cells into the environment, elimination rates would be comparable to those for non-recombinant yeast strains.

Cloning and characterization of OSF-3, a new member of the MER5 family, expressed in mouse osteoblastic cells.

A cDNA library prepared from the mouse osteoblastic cell line, MC3T3-E1, was screened for the presence of specifically expressed genes by employing subtraction hybridization/differential screening methods. A cDNA clone was identified and sequenced, encoding a protein designated as osteoblast specific factor 3 (OSF-3) comprising 199 amino acids. RNA dot blot analysis indicated weak OSF-3 expression in thymus, spleen, brain, lung, testis, and heart, and high expression in kidney and liver. A homology search of an amino acid sequence database revealed a strong relationship of OSF-3 to the MER5 (gene preferentially expression in murine erythroleukemia cells) protein and human pag (proliferation associated gene) product. This indicates that OSF-3 plays an intrinsic role in the proliferation and/or differentiation of bone cells.

Molecular cloning and characterization of OB-cadherin, a new member of cadherin family expressed in osteoblasts.

The preferential screening of cDNA libraries derived from the mouse osteoblastic cell line MC3T3-E1 has yielded a cDNA clone encoding 796 amino acids including the typical 24-residue-long signal sequence. The predicted amino acid sequence revealed that this protein constitutes a new member of the cadherin family. We propose the name OB-cadherin (OB for osteoblast) for this new protein. RNA analyses revealed that the OB-cadherin gene is selectively expressed in osteoblastic cell lines, precursor cell lines of osteoblasts, and primary osteoblastic cells from calvaria, as well as in lung, testis, and brain tissues at low levels. Transfection of OB-cadherin cDNA into L cells resulted in the acquisition of the Ca(2+)-dependent adhesive property. Two different forms of the human OB-cadherin cDNA were subsequently cloned; one is a counterpart of mouse OB-cadherin gene, and the other encodes a protein with a truncated cytoplasmic domain. The results suggest that the newly found OB-cadherin might have an important role in bone formation.

Antibody recognition of the recombinant human nuclear antigens RNP 70 kD, SS-A, SS-B, Sm-B, and Sm-D by autoimmune sera.

Five human nuclear antigens, RNP 70 kD, SS-A, SS-B, Sm-B and Sm-D, were produced in E. coli using the expression vector pSEM. cDNAs encoding these antigens were ligated to a truncated lacZ' gene of the vector and the beta-galactosidase fusion proteins were efficiently expressed as intracellular inclusion bodies after isopropyl-beta-thiogalactopyranoside induction. The antibody reactivities of these fusion proteins were evaluated by Western blot and by ELISA employing panel sera from patients with autoimmune diseases such as systemic lupus erythematosus, Sjögren's syndrome or mixed connective tissue disease. The three fusion proteins, RNP 70 kD, SS-B, and Sm-B, showed good reactivities in both systems, whereas the other two fusion proteins, SS-A and Sm-D, showed poor and no reactivity in both systems, respectively. It can be concluded that RNP 70 kD, SS-B and Sm-B recombinant antigens are useful reagents for the differential diagnosis of the autoimmune diseases.

Specific targeting and killing activities of anti-P-glycoprotein monoclonal antibody MRK16 directed against intrinsically multidrug-resistant human colorectal carcinoma cell lines in the nude mouse model.

Anti-P-glycoprotein (P-gp) monoclonal antibody, MRK16, and its F(ab')2 fragment were evaluated for its therapeutic efficacy to P-gp-mediated multidrug resistant human colorectal carcinoma cell lines in a nude mouse model. In a blood clearance experiment, 125I-labeled MRK16 had a half-life (16 h) 7 times longer than its F(ab')2 fragment (half-life of 1.8 h) in circulation in nude mice, and approximately 16 and 5% of MRK16 were retained on days 10 and 20 after injection, respectively. In biodistribution experiments using nude mice bearing HCT-15, an intrinsically resistant cell line, 125I-labeled MRK16 accumulated at the tumor site significantly higher than its F(ab')2 fragment as revealed by the percentage of injected dose/g of tissue values (7.4 versus 0.6%) on day 3 after injection. In contrast, the tissue to blood ratio at the tumor site of the MRK16 was significantly lower than that of its F(ab')2 fragment (1.2 versus 10.5). Specific targeting of the MRK16 F(ab')2 fragment to the P-gp-positive tumor (HCT-15) but not to the P-gp-negative tumor (COLO 205) was observed in the nude mice bearing both tumors. In the therapeutic efficacy tests, when administered i.v. 3 times on days 1, 4, and 7 after tumor s.c. inoculation, MRK16 alone showed the significant inhibition of tumor growth of P-gp-positive cell lines, HCT-15, DLD-1, SW480, and SW1417 in contrast to cases of P-gp-negative cell lines, COLO 205 and KM20L2. This inhibitory effect of MRK16 was enhanced in combination with Adriamycin, which alone hardly inhibited the tumor growth. However, MRK16 F(ab')2 fragment alone, even at 1 mg/mouse, had little inhibitory effect on the growth of HCT-15 in the same treatment schedule. When administered at early palpable stage, the degree of HCT-15 tumor growth suppression depended on the number of MRK16 injections. At more progressed stages, treatment with MRK16 alone showed little antitumor activity but when combined with Adriamycin resulted in significant suppression of tumor growth. The present results suggest that MRK16 may be useful for in vivo immunoscintigraphy and immunotherapy of multidrug-resistant colorectal carcinoma.

Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation.

A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: Beneficial effect of sucrose and raffinose on their cryosurvival rate.

Survival of mouse 2-cell embryos was evaluated after exposure to 1.38, 2.75 or 5.5 M single cryoprotectants [dimethylsulphoxide (DMSO), acetamide (Ac) and propylene glycol (PG)], components frequently utilized as a vitrification solution, for 0.5, 1, 2 and 10 minutes at room temperature prior to vitrification. More than 80 % of the treated embryos developed to normal blastocysts in culture, after exposure to 1.38-2.75 M of each reagent for 0.5 minutes, although Ac tended to provide with have a deleterious effect on their survival. When the embryos were vitrified with solutions containing DP (2.75 M DMSO and 2.75 M PG) plus 0, 0.5 and 1.0 M Ac after a 0.5-minute exposure, their in vitro survival rates to the blastocysts were 44, 41 and 37%, respectively, showing no significant difference among them (x(2)=0.1-0.6, P>0.05). This indicates that the presence of Ac is not always needed for vitrifying mouse 2-cell embryos. Embryos, that had been vitrified with DP solution supplemented with 1.0 M sucrose (DPS) after a 0.5- minute exposure, exhibited significantly higher in vitro survival rate (82%) than those vitrified with DP (44%) (x(2)=23.4, P<0.001). Similar high survival rate (81%) was obtained when they were vitrified with DP plus 0.16 M raffinose (DPR) (x(2)=28.3, P<0.001). In vivo survival of embryos vitrified with DPS or DPR after a 0.5-minute exposure was both 49%, and there was no significant difference comparing to the unvitrified control group (60%). This method is rapid, efficient and reliable, and thus may be of practical use for cryopreserving mouse 2-cell embryos.

cDNA expression cloning of the 85-kDa protein overexpressed in adriamycin-resistant cells.

An 85-kDa cell surface protein recognized by monoclonal antibody MRK-20 was identified in adriamycin-resistant tumor cells. The expression of the 85-kDa protein has recently been reported to be associated with the differentiation of hematopoietic cells. However, the primary structure of the 85-kDa protein has not been determined. To clarify its primary structure, we carried out cDNA expression cloning of the 85-kDa protein with monoclonal antibody MRK-20. We found that the 85-kDa protein is identical to CD36 (GP VI), but at least two species of transcript exist in the tumor cells and one of these transcripts has a novel sequence at the 3'-region. The transcript with the novel sequence at the 3'-region was found to be expressed during the differentiation of hematopoietic cells.

Gene introduction into mouse blastocysts via "pricking".

It is a well-known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation 'pricking' technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid-gestation stage were obtained. Twenty-two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)-Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli beta-galactosidase (beta-gal) gene to a mouse metallothionein-I (MT-I) promoter and assessed for beta-gal activity histochemically after 1 and 5 days of culture in the presence of 1 microM CdCl2, at least 65% of the embryos exhibited beta-gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique.