Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes

Frank Martiniuk, Angel Pellicer, Mark F. Mehler, Rochelle Hirschhorn

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neo and analyzed 121 NeoRclones for expression of 15 human "housekeeping" enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 NeoRclones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 NeoRclones and found 25 clones expressing human GAA, as determined by rocket immuno-electrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo -derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector.

Original languageEnglish (US)
Pages (from-to)1-12
Number of pages12
JournalSomatic Cell and Molecular Genetics
Volume12
Issue number1
DOIs
StatePublished - Jan 1986

Fingerprint

Enzymes
Housekeeping
alpha-Glucosidases
proline dipeptidase
Electrophoresis
Clone Cells
Alu Elements
Phosphoglucomutase
Bacterial Genes
3T3 Cells
Acids
DNA
Growth
Genes

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes. / Martiniuk, Frank; Pellicer, Angel; Mehler, Mark F.; Hirschhorn, Rochelle.

In: Somatic Cell and Molecular Genetics, Vol. 12, No. 1, 01.1986, p. 1-12.

Research output: Contribution to journalArticle

Martiniuk, Frank ; Pellicer, Angel ; Mehler, Mark F. ; Hirschhorn, Rochelle. / Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes. In: Somatic Cell and Molecular Genetics. 1986 ; Vol. 12, No. 1. pp. 1-12.
@article{66983bedb4e94f6bb1632f2166afac23,
title = "Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes",
abstract = "We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neo and analyzed 121 NeoRclones for expression of 15 human {"}housekeeping{"} enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 NeoRclones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 NeoRclones and found 25 clones expressing human GAA, as determined by rocket immuno-electrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo -derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector.",
author = "Frank Martiniuk and Angel Pellicer and Mehler, {Mark F.} and Rochelle Hirschhorn",
year = "1986",
month = "1",
doi = "10.1007/BF01560722",
language = "English (US)",
volume = "12",
pages = "1--12",
journal = "Somatic Cell and Molecular Genetics",
issn = "0740-7750",
publisher = "Springer GmbH & Co, Auslieferungs-Gesellschaf",
number = "1",

}

TY - JOUR

T1 - Detection, frequency, and stability of cotransformants expressing nonselectable human enzymes

AU - Martiniuk, Frank

AU - Pellicer, Angel

AU - Mehler, Mark F.

AU - Hirschhorn, Rochelle

PY - 1986/1

Y1 - 1986/1

N2 - We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neo and analyzed 121 NeoRclones for expression of 15 human "housekeeping" enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 NeoRclones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 NeoRclones and found 25 clones expressing human GAA, as determined by rocket immuno-electrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo -derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector.

AB - We cotransformed mouse 3T3 cells with total genomic human DNA and the dominant selectable bacterial gene Neo and analyzed 121 NeoRclones for expression of 15 human "housekeeping" enzymes which can be distinguished from their murine homologs. The estimated frequency of expression of unlinked human genes was 1 in 360 NeoRclones and at least three different human enzymes (peptidase D, phosphoglucomutase 1, and acid alpha glucosidase) were detected. We further examined the frequency and stability of cotransformation for one of these enzymes, acid alpha glucosidase (GAA). We tested approximately 4000 NeoRclones and found 25 clones expressing human GAA, as determined by rocket immuno-electrophoresis (RIE) specific for human GAA. Transformants progressively became negative on continued growth and retesting by RIE, with only two clones still expressing GAA at the eighth testing. This apparent loss of expression was not due to nonclonality of the original isolates. In one subclone examined, loss of expression was accompanied by loss of both Neo -derived pBR322 and human Alu repetitive sequence DNA. Thus, under the conditions utilized, cotransformants expressing homomeric housekeeping enzymes were found at relatively high frequency but were progressively lost even under conditions selective for expression of the dominant vector.

UR - http://www.scopus.com/inward/record.url?scp=0022641215&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0022641215&partnerID=8YFLogxK

U2 - 10.1007/BF01560722

DO - 10.1007/BF01560722

M3 - Article

VL - 12

SP - 1

EP - 12

JO - Somatic Cell and Molecular Genetics

JF - Somatic Cell and Molecular Genetics

SN - 0740-7750

IS - 1

ER -